( PDF ) Caracterização de protease e lipase de pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite

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MAURILIO LOPES MARTINS

CARACTERIZAÇÃO DE PROTEASE E LIPASE DE Pseudomonas

fluorescens E QUORUM SENSING EM BACTÉRIAS PSICROTRÓFICAS

ISOLADAS DE LEITE

Tese apresentada à Universidade Federal de

Viçosa, como parte das exigências do Programa

de Pós-Graduação em Microbiologia Agrícola,

para obtenção do título de Doctor Scientiae.

VIÇOSA

MINAS GERAIS - BRASIL

2007

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MAURILIO LOPES MARTINS

CARACTERIZAÇÃO DE PROTEASE E LIPASE DE Pseudomonas

fluorescens E QUORUM SENSING EM BACTÉRIAS PSICROTRÓFICAS

ISOLADAS DE LEITE

Tese apresentada à Universidade Federal de

Viçosa, como parte das exigências do Programa

de Pós-Graduação em Microbiologia Agrícola,

para obtenção do título de Doctor Scientiae.

APROVADA: 23 de março de 2007.

Prof

. Elza Fernandes Araújo Prof. Hilário Cuquetto Mantovani

(Co-orientadora) (Co-orientador)

Prof

. Andréa de Oliveira Barros Ribon

. Anna Katharina Maria Riedel

Prof

. Maria Cristina Dantas Vanetti

(Orientadora)

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À minha querida esposa Eliane e

aos meus queridos pais Geralda e Bolivar,

Dedico.

iii

AGRADECIMENTOS

Aos meus pais, Bolivar e Geralda, pelo amor, apoio, incentivo, pela

dedicação e pelo esforço que tornaram possível minha formação.

A Eliane, pela presença insubstituível em minha vida, e pelo carinho,

companheirismo e amor que foram indispensáveis a cada momento e amenizaram as

dificuldades e obstáculos encontrados no caminho.

À Universidade Federal de Viçosa pelos anos dedicados a minha formação e

pela estrutura que tornou possível o meu desenvolvimento profissional e intelectual.

Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), pelo auxílio financeiro e pela oportunidade de realização dos experimentos

de tese na Universidade de Zurique.

À professora Maria Cristina Dantas Vanetti, pela orientação e pelo estímulo

que tornaram possível o desenvolvimento deste trabalho, e pela amizade.

À Drª. Anna Katharina Maria Riedel, pela excelente convivência, pelo auxílio

e incentivo, pela amizade, dedicação, e pelas valiosas sugestões.

Ao professor Dr. Leo Eberl, por me acolher de braços abertos no Laboratório

de Microbiologia da Universidade de Zurique e pelo apoio que tornou possível a

realização deste trabalho.

Aos amigos do Laboratório de Microbiologia da Universidade de Zurique

Julia, Silvia, Paula, Sue, Susane, Jasmim, Celine, Urs, e Manoel, pela excelente

convivência e pelo companherismo.

Ao professor Dr. Jorge Leitão da Universidade de Lisboa, pela amizade, pelo

auxílio e pelos conselhos que foram fundamentais para minha adaptação e ao

desenvolvimento dos experimentos no Laboratório de Microbiologia da

Universidade de Zurique.

À Drª. Judith Blom, pelo auxílio nos ensaios de caracterização de acil

homoserina lactonas.

À Drª. Verena Thiel e ao professor Dr. Stefan Schulz da Universidade

Técnica de Braunschweig, Alemanha, pela caracterização das moléculas presentes

nos extratos obtidos a partir de meio TYEP.

À professora Elza Fernandes de Araújo, pelas críticas e valiosas sugestões na

discussão e organização deste trabalho, e pela disponibilidade do Laboratório de

Genética Molecular e de Microrganismos.

Aos professores Hilário Cuquetto Mantovani, Andréa de Oliveira Barros

Ribon e Célia Alencar de Moraes pelas sugestões.

A todos os professores do Departamento de Microbiologia da UFV, pelos

ensinamentos.

Aos professores da área de Laticínios do Departamento de Tecnologia de

Alimentos da UFV, pela minha formação básica.

A todos os amigos, companheiros de curso e de laboratório, representados por

Adriana Ponce, Agenor, Ana Andréa, Ana Diolina, Andréa, Bete, Cristiane, Cláudia

Lúcia, Eliane, Eliseth, Esther, Fernanda, Flávia, Francis, Gildete, Janaína, João

Batista, Jorge, Marcelão, Marília, Renata, Rodrigo, Selma, Simone Quintão,

Uelinton, Wanessa, Wendel e tantos outros aqui não citados, pela amizade, pelo

apoio e pela agradável convivência, fundamentais para que eu pudesse crescer tanto

pessoal quanto profissionalmente.

Ao grande amigo Maurício de Oliveira Leite, funcionário do Departamento

de Tecnologia de Alimentos, pelas dicussões, pelo estudo em grupo, pela

convivência, pelo apoio e pela consideração.

A Eliseth, pela grande amizade e consideração, pelos estudos em grupo, por

me influenciar a trabalhar com quorum sensing e pelos momentos de recreação.

À Drª. Cláudia Lúcia de Oliveira Pinto, pelo isolamento das culturas

utilizadas neste trabalho, pelo incentivo, pela amizade e consideração.

Ao meu tio, padrinho e amigo Luís Tadeu Lopes, pelo apoio, incentivo, pelos

conselhos e pela presença em todos os momentos de minha vida.

A todos os funcionários do Departamento de Microbiologia e do BIOAGRO,

pela amizade e pelos serviços prestados.

Aos funcionários Adriana Leandro, Pablo, José Carlos e Sr. Paulo, pela

simpatia e atenção constantes.

A todas as pessoas que, de alguma forma, contribuíram para a realização

deste trabalho e para o meu crescimento pessoal e profissional.

A Deus, por último, mas acima de tudo e de todos, por ter me proporcionado

inteligência e perseverança para vencer os obstáculos, e pela presença insubstituível

em cada instante de minha vida.

BIOGRAFIA

MAURILIO LOPES MARTINS, filho de Bolivar Martins de Miranda e

Geralda Lopes Martins, nasceu em Rio Pomba, Minas Gerais, no dia 19 de julho de

1974.

Em setembro de 1996, graduou-se como Tecnólogo em Laticínios pela

Universidade Federal de Viçosa (UFV), em Viçosa-MG. No período de 1996 a 2000,

foi funcionário do Departamento de Garantia de Qualidade da Cooperativa Central

de Laticínios do Estado de São Paulo (Leite Paulista).

Em outubro de 2000, retornou para a UFV onde iniciou o curso de graduação

em Ciência e Tecnologia de Laticínios, concluindo-o em 2001. Nesse mesmo ano,

iniciou o curso de Mestrado em Microbiologia Agrícola na UFV, concentrando seus

estudos na área de Microbiologia de Alimentos, defendendo tese em 26 de fevereiro

de 2003.

Em março do mesmo ano, iniciou o curso de Doutorado em Microbiologia

Agrícola na mesma área e instituição. Em 2005, obteve uma bolsa de doutorado

sanduíche do CNPq e desenvolveu os experimentos de tese na Universidade de

Zurique, Suíça. Em novembro de 2006, retornou ao Brasil e em março de 2007

concluiu sua tese na UFV.

vii

SUMÁRIO

LISTA DE ABREVIATURAS...............................................................................xiii

RESUMO.................................................................................................................xvi

ABSTRACT...........................................................................................................xviii

INTRODUÇÃO GERAL...........................................................................................1

CHAPTER 1

LITERATURE REVIEW..........................................................................................3

1.1. Psychrotrophic bacteria in milk ........................................................................ 3

1.2. Relevance of spoilage enzymes produced by psychrotrophic bacteria in milk 4

1.3. Regulation of spoilage enzymes expression by P. fluorescens......................... 6

1.4. The mechanism of quorum sensing ..................................................................9

1.4.1. Biosynthesis and characterization of the signal molecules...................... 10

1.4.1.1. Acyl-homoserine lactones (AHLs) ....................................................... 10

1.4.1.2. Quinolones ............................................................................................ 11

1.4.1.3. Diketopiperazines (DKP)...................................................................... 13

1.4.1.4. Furanosyl borate diester: auto-inducer two (AI-2)................................ 14

1.4.1.5. Oligopeptides ........................................................................................ 15

1.4.2. Detection of signal molecules.................................................................. 16

1.4.3. Cross-communication .............................................................................. 17

1.4.4. Control of the quorum sensing system..................................................... 17

viii

1.4.5. Relevance of quorum sensing mechanism to the food industry............... 19

1.5. REFERENCES..................................................................................................20

CHAPTER 2

OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF

MILK-DETERIORATING HYDROLYTIC EXOENZYMES PRODUCED

BY Pseudomonas fluorescens ..................................................................................30

2.1. INTRODUCTION.............................................................................................30

2.2. MATERIAL AND METHODS........................................................................32

2.2.1. Bacterial strains and plasmids...................................................................... 32

2.2.2. Growth conditions........................................................................................ 33

2.2.3. Protein quantifiction and enzyme assays ..................................................... 33

2.2.3.1. Protein quantification............................................................................ 33

2.2.3.2. Protease assay........................................................................................ 33

2.2.3.3. Lipase assay .......................................................................................... 34

2.2.4. SDS-PAGE and zymograms ........................................................................ 34

2.2.5. Identification of proteins by mass spectrometry .......................................... 35

2.2.6. DNA manipulation, PCR reaction and sequencing...................................... 36

2.2.6.1. DNA manipulations .............................................................................. 36

2.2.6.2. Amplification of the protease and lipase genes by PCR....................... 36

2.2.6.3. Sequencing of the protease and lipase genes ........................................ 37

2.2.7. Cloning, heterologous expression and purification of P. fluorescens 041

protease and lipase ...................................................................................... 38

2.2.8. Biochemical characterization of purified enzymes...................................... 39

2.2.8.1. Temperature optimum........................................................................... 39

2.2.8.2. pH optimum .......................................................................................... 39

2.2.8.3. Heat stability ......................................................................................... 39

2.2.8.4. Metal ions.............................................................................................. 39

2.2.8.5. Protease inhibitors................................................................................. 40

2.2.9. Substrate specificity ..................................................................................... 40

2.2.9.1. Protease ................................................................................................. 40

2.2.9.2. Lipase.................................................................................................... 40

2.3. RESULTS AND DISCUSSION .......................................................................41

2.3.1. Milk-deteriorating hydrolytic activities of P. fluorescens ........................... 41

2.3.2. Cloning and sequencing of protease and lipase genes ................................. 43

2.3.3. Overexpression and purification of AprX and LipM................................... 44

2.3.4. Biochemical characterization of AprX and LipM........................................ 45

2.4. CONCLUSIONS ...............................................................................................53

2.5. REFERENCES..................................................................................................54

CHAPTER 3

INVESTIGATION OF QUORUM SENSING IN STRAINS OF Pseudomonas

fluorescens ISOLATED FROM REFRIGERATED RAW MILK......................58

3.1. INTRODUCTION.............................................................................................58

3.2. MATERIAL AND METHODS........................................................................61

3.2.1. Bacterial strains and growth conditions....................................................... 61

3.2.2. Detection and quantification of signal molecules........................................ 63

3.2.3. Extraction of quorum sensing signal from supernatants.............................. 64

3.2.4. Detection of signal molecules in supernatant of P. fluorescens................... 65

3.2.5. Detection of signal molecules using Thin Layer Chromatography (TLC) .. 65

3.2.6. DNA manipulations, PCR reactions and sequencing................................... 66

3.2.6.1. DNA manipulations .............................................................................. 66

3.2.6.2. Amplification of the AHL synthase (phzI and mupI) genes of P.

fluorescens by PCR...............................................................................

3.2.6.3. Sequencing of the AHL synthase genes................................................ 67

3.2.7. Evaluation of P. fluorescens resistance against different antibiotics and

tellurite ........................................................................................................

3.2.8. Cloning of the gentamicin-3-acetyltransferase gene on broad-host-range

expression vector......................................................................................... 67

3.2.9. Conjugative plasmid transfer ....................................................................... 67

3.2.10. Phenotypic characterization of wild type and transconjugant strains........ 68

3.2.11. Identification of signal molecules by mass spectrometry .......................... 69

3.2.12. Detection of AI-2 in supernatant of LB medium inoculated with P.

fluorescens ..................................................................................................

3.3. RESULTS AND DISCUSSION .......................................................................70

3.3.1. Detection of signal molecules produced by P. fluorescens.......................... 70

3.3.2. Detection of bioluminescence induced by P. fluorescens............................ 72

3.3.3. Supplementation of LB inoculated with E. coli MT102 pSB403 with

extracts obtained from different media ....................................................... 73

3.3.4. Detection of signal molecules using TLC.................................................... 74

3.3.5. Amplification and sequencing of phzI and mupI genes by PCR.................. 77

3.3.6. Resistance of P. fluorescens against some antibiotics and selective agent.. 78

3.3.7. Cloning of gentamicin-3-acetyltransferase gene in pMLBAD-aiiA-Trm

and

mobilization to P. fluorescens 07A and 041...............................................

3.3.8. Phenotypic characteristics of P. fluorescens wild type and trans

conjugants ................................................................................................... 81

3.3.8.1. Biofilm .................................................................................................. 81

3.3.8.2. Swarming motility................................................................................. 82

3.3.8.3. Extracellular protease............................................................................ 83

3.3.9. Chemical characterization of signal molecules produced by

P. fluorescens 07A into TYEP medium...................................................... 84

3.3.10. Detection of AI-2 ....................................................................................... 85

3.4. CONCLUSIONS ...............................................................................................87

3.5. REFERENCES..................................................................................................88

CHAPTER 4

QUORUM SENSING IN PSYCHROTROPHIC STRAINS ISOLATED

FROM REFRIGERATED RAW MILK ...............................................................

4.1. INTRODUCTION.............................................................................................94

4.2. MATERIAL AND METHODS........................................................................97

4.2.1. Strains and growth conditions...................................................................... 97

4.2.2. Amplification and sequencing of 16S rDNA from psychrotrophic strains.. 99

4.2.3. Milk spoilage potential and production of exoenzymes by psychrotrophic

strains .......................................................................................................... 99

4.2.4. Detection and quantification of AHL......................................................... 100

4.2.5. Extraction of AHLs from supernatants ...................................................... 100

4.2.6. Detection of AHL using Thin Layer Chromatography (TLC)................... 101

4.2.7. LC-MS analysis of AHL extracts from bacterial supernatants.................. 101

4.2.8. Resistance of psychrotrophic strains against different antimicrobials....... 101

4.2.9. Conjugative plasmid transfer and confirmation of identity of

transconjugant strains................................................................................ 102

4.2.10. Phenotypic characterization of wild type and transconjugant strains...... 102

4.2.11. Detection and sequencing of a native plasmid from H. alvei 068

and 071...................................................................................................... 103

4.2.12. DNA manipulations, PCR reactions and sequencing of halI and halR

genes..........................................................................................................

103

4.2.13. Cloning and heterologous expression of AHL synthase (halI) of H.

alvei 068 in pQE-30Xa.............................................................................. 104

4.2.14. Detection, extraction, and characterization of AHLs encoded by halI.... 105

4.2.15. Detection of AI-2 in supernatant of LB medium inoculated with

psychrotrophic strains ............................................................................... 106

4.2.16. Pathogenesis of psychrotrophic strains against Caenorhabditis

elegans ...................................................................................................... 106

4.2.16.1. Maintenance and cultivation of C. elegans....................................... 106

4.2.16.2. Egg preparation of C. elegans........................................................... 106

4.2.16.3. Nematode assays............................................................................... 107

4.3. RESULTS AND DISCUSSION .....................................................................108

4.3.1. Confirmation of identity of psychrotrophic strains isolated from cooled

raw milk .................................................................................................... 108

4.3.2. Spoilage potential and production of exoenzymes by psychrotrophic

strains isolated from cooled raw milk.......................................................

109

4.3.2.1. Potential to spoil milk samples ........................................................... 109

4.3.2.2. Production of extracellular enzymes................................................... 111

4.3.3. Detection of AHL molecules produced by Enterobacter sp., H. alvei,

and A. hydrophila...................................................................................... 114

4.3.4. Characterization of AHL molecules using TLC ........................................ 115

4.3.5. Characterization of AHL molecules by liquid chromatography-mass

spectrometry (LC-MS).............................................................................. 117

4.3.6. Resistance of psychrotrophic strains against antibiotics and tellurite........ 126

xii

4.3.7. Quorum quenching mechanism in Enterobacter and H. alvei................... 127

4.3.7.1. Mobilization of pBHR1-aiiA .............................................................. 127

4.3.7.2. AHL production by transconjugant strains ......................................... 128

4.3.7.3. Proteolytic activity of wild type and transconjugants......................... 129

4.3.7.4. Influence of quorum quenching mechanism in expression of

extracellular proteins by Enterobacter sp. 067 ................................... 132

4.3.8. Native plasmid of H. alvei ......................................................................... 133

4.3.9. Amplification of AHL synthase (halI) and AHL receptor (halR) genes

by PCR ......................................................................................................

135

4.3.10. Sequencing and overexpression of halI in E. coli XL1-Blue................... 137

4.3.11. Detection of AHL molecules produced by E. coli

XL1-Blue pQE-30Xa-halI......................................................................... 139

4.3.11.1. Cross-streak assay............................................................................. 139

4.3.11.2. Thin layer chromatography assay ..................................................... 139

4.3.11.3. Chemical characterization of AHL molecules by LC-MS................ 141

4.3.12. Detection of auto-inducer two (AI-2) ...................................................... 142

4.3.13. Pathogenesis against Caenorhabditis elegans ......................................... 142

4.4. CONCLUSIONS .............................................................................................145

4.5. REFERENCES................................................................................................146

APPENDICES........................................................................................................152

xiii

LISTA DE ABREVIATURAS

A136 – Estirpe de Agrobacterium tumefaciens biosensora de AHL

AB Agrobacterium – Meio mínimo para crescimento de estirpes de Agrobacterium

ABC – Meio mínimo suplementado com citrato

ABG – Meio mínimo suplementado com glicose

AB Vibrio – Meio mínimo para crescimento de estirpes de Vibrio

ACN – Acetonitrila

ACP – Proteína carreadora de acil

AHL – Homoserina lactona acilada

AI-2 – autoindutor dois

AI-3 – autoindutor três

AiiA – Enzima lactonase, cliva o anel lactona da molécula de AHL

AprX – Metalloprotease alcalina de Pseudomonas fluorescens

BHL – N-butanoil-DL-homoserina lactona

C4-HSL – N-butanoil-DL-homoserina lactona

C5-HSL – N-pentanoil-DL-homoserina lactona

C6-HSL – N-hexanoil-DL-homoserina lactona

C8-HSL – N-octanoil-DL-homoserina lactona

C10-HSL – N-decanoil-DL-homoserina lactona

CFU/ml – Unidade formadora de colônia por mililitro

CV026 – Estirpe mutante de Chromobacterium violaceum biosensora de AHL

DHL – N-dodecanoil-DL-homoserina lactona (C12HSL)

xiv

DKP – Dicetopiperazina

DNA – Ácido desoxirribonucléico

DPD – 4,5-dihidroxi-2,3-pentanodieno

DTT – Ditiotreitol

dYT – Meio triptona, extrato de levedura, NaCl e glicose

EDTA – ácido etilenodiaminotetracético

ESI-MS – Ionização por electrospray – espectrometria de massa

FUR – Regulador de absorção de ferro

GC – Cromatografia gasosa

GC-MS – Cromatografia gasosa acoplada a espectrometria de massa

GMP – Boas práticas de fabricação

HHL – N-hexanoil-DL-homoserina lactona (C6-HSL)

HHQ – 2-heptil-4-quinolona

HPLC – Cromatografia líquida de alta performance

HSL – Homoserina lactona

HTST – Alta temperatura e curto tempo ou pasteurização rápida

IPTG – Isopropil-β-D-tiogalactopiranosideo

KYC55 – Estirpe de Agrobacterium tumefaciens biosensora de AHL

LB – Meio Luria Bertani

LC-MS – Cromatografia líquida acoplada a espectrometria de massa

LipM – Lipase de Pseudomonas fluorescens

LTLT – Baixa temperatura e longo tempo ou pasteurização lenta

MALDI-TOF – Matrix assisted laser desorption ionization – time of flight

MCS – Sítio de clonagem múltipla

MMS – Meio mínimo de sais

MS - Espectrometria de massa

NGMI e NGMII – Meio para crescimento de nematóide

NTL4 – Estirpe de Agrobacterium tumefaciens biosensora de AHL

OHHL – N-3-oxohexanoil-L-homoserina lactona (3-oxo-C6-HSL)

OHL - N-octanoil-DL-homoserina lactona

ORF – Quadro de leitura aberto

PCR – Reação de polimerização em cadeia

pMLM – plasmídeo nativo de Hafnia alvei 068 e 071

PMSF – Fenilmetilsulfonil fluoride

PQS – 2-heptil-3-hidroxi-4-quinolona ou quorum sensing em Pseudomonas

QS – quorum sensing

RPM – Rotação por minuto

SAH – S-adenosil homosisteína

SAM – S-adenosil metionina

SDS - Dodecil sulfato de sódio

SDS-PAGE – Gel de poliacrilamida-dodecil sulfato de sódio

SRH – S-ribosil homosisteína

TCA – Ácido tricloroacético

TLC – Cromatografia em camada fina

TYEP – Meio triptona, extrato de levedura e fosfato

UHT – Ultra-alta temperatura

X-gal – 5-bromo-4-cloro-3-indolil-β-D-galactopiranosídeo

3-hidroxi-C4-HSL – N-3-hidroxibutanoil-DL-homoserina lactona

3-hidroxi-C12-HSL – N-3-hidroxidodecanoil-DL-homoserina lactona

3-oxo-C6-HSL – 3-oxohexanoil-DL-homoserina lactona

3-oxo-C8-HSL – N-3-oxooctanoil-DL-homoserina lactona

3-oxo-C10-HSL – N-3-oxodecanoil-DL-homoserina lactona

3-oxo-C12-HSL – 3-oxododecanoil-DL-homoserina lactona

xvi

RESUMO

MARTINS, Maurilio Lopes, D.Sc., Universidade Federal de Viçosa, março de 2007.

Caracterização de protease e lipase de Pseudomonas fluorescens e quorum

sensing em bactérias psicrotróficas isoladas de leite. Orientadora: Maria

Cristina Dantas Vanetti. Co-orientadores: Elza Fernandes de Araújo, Hilário

Cuquetto Mantovani e Célia Alencar de Moraes.

Protease e lipase produzidas por Pseudomonas fluorescens foram purificadas

e caracterizadas. Os genes aprX e lipM foram clonados, seqüenciados, e expressos

em Escherichia coli e apresentaram alta identidade com as seqüências disponíveis no

banco de dados. A massa molecular deduzida de ambas as enzimas foi de 50 kDa.

Foi verificado que cálcio é essencial para as atividades enzimáticas, uma vez que

quando este íon não foi adicionado à solução de diálise nenhuma atividade foi

encontrada. A protease foi ativa em ampla faixa de pH, apresentou temperatura ótima

de 37 °C, e maior atividade foi verificada sobre caseína e gelatina. Maior estabilidade

térmica da enzima foi a 75 °C por 20 s. A lipase foi mais ativa a 25 °C e em pH

próximo de 7,5 e p-nitrofenil-palmitato foi o substrato preferencial. Tratamentos

térmicos de 65 °C por 30 min e de 75 °C por 1 min reduziram sua atividade para

13,2% e 25,4%, respectivamente. O mecanismo de quorum sensing (QS) em P.

fluorescens foi estudado e verificou-se que as estirpes avaliadas, apesar de induzirem

Agrobacterium tumefaciens NTL4 e A136, não produziram acil homoserinas

lactonas (AHLs). Entretanto, a produção de auto-indutor dois (AI-2) foi detectada e a

xvii

presença de dicetopiperazinas (DKPs) nos extratos químicos obtidos a partir de meio

TYEP inoculado com P. fluorescens foi constatada. O rDNA 16S de seis bactérias

gram-negativas isoladas de leite cru foi seqüenciado e o isolado 039 foi identificado

como Pantoea sp., 059, 068 e 071 foram identificados como Hafnia alvei, 067 como

Enterobacter sp. e 099 como Aeromonas hydrophila. Verificou-se que esses isolados

apresentam diferenças no potencial deteriorador, na resistência a antibióticos e, após

ensaios de estria cruzada em superfície de meio sólido para detecção de AHLs,

constatou-se que somente Pantoea sp. não foi capaz de induzir nenhuma das estirpes

monitoras utilizadas. Os ensaios de cromatografia em camada fina e a caracterização

química dos extratos por espectrometria de massa confirmaram que essas bactérias

produzem diferentes AHLs. O mecanismo de inibição de quorum foi utilizado e a

enzima lactonase foi expressa em Enterobacter sp. transconjugante a qual foi incapaz

de acumular AHLs em sobrenadante de caldo LB. O transconjugante se mostrou

mais proteolítico do que a estirpe selvagem, indicando que o mecanismo de QS

regula negativamente a atividade proteolítica neste isolado. Das 32 enzimas de

restrição utilizadas para restringir o plasmídeo nativo (pMLM) presente nos isolados

de H. alvei 068 e 071, apenas DdeI, HinfI, MspI, e RsaI foram efetivas. Além disso,

não foi possível expressar a enzima lactonase em H. alvei 068 e 071 transconjugantes

o que impossibilitou a avaliação da influência do mecanismo de QS sobre a atividade

proteolítica desses isolados. O gene halI, que codifica a sintase de AHLs produzidas

por estirpes de H. alvei, foi identificado nos isolados 059, 067, 068 e 071. Esse gene

foi clonado, seqüenciado e expresso em E. coli e verificou-se que codifica uma

sintase responsável pela produção de N-hexanoil-DL-homoserina lactona (C6-HSL)

e N-3-oxohexanoil-L-homoserina lactona (3-oxo-C6-HSL). Das seis estirpes

psicrotróficas proteolíticas avaliadas, apenas A. hydrophila foi capaz de produzir

quitinase, AI-2 e de ser patogênica contra Caenorhabditis elegans.

xviii

ABSTRACT

MARTINS, Maurilio Lopes, D.Sc., Federal University of Viçosa, March 2007.

Characterization of protease and lipase from Pseudomonas fluorescens and

quorum sensing in psychrotrophic bacteria isolated from milk. Adviser:

Maria Cristina Dantas Vanetti. Co-Advisers: Elza Fernandes de Araújo, Hilário

Cuquetto Mantovani and Célia Alencar de Moraes.

A protease and a lipase produced by Pseudomonas fluorescens were purified

and characterized. The aprX and lipM genes were cloned, sequenced, and expressed

in Escherichia coli. These genes presented high identity with the sequences available

in the GenBank. The molecular mass of both enzymes were 50 kDa. It was verified

that calcium is essential to the enzymatic activities since when this ion was not added

into the dialysis solution no activity was found. The protease was active in a large

range of pH, had highest activity against casein and gelatin, and its temperature

optimum was at 37 °C. Besides, this enzyme showed the highest thermal stability at

75 °C for 20 s. In contrast to the protease, the temperature optimum for the lipase

was 25 °C and the pH optimum was close to 7.5. This enzyme showed the highest

activity against p-nitrophenyl-palmitate, and the thermal treatments of 65 °C for

30 min and 75 °C for 1 min reduced its activity to 13.2% and 25.4%, respectively.

The mechanism of quorum sensing (QS) was studied in P. fluorescens and it was

verified that although the strains evaluated induced Agrobacterium tumefaciens

NTL4 and A136, they do not produce acyl-homoserine lactones (AHLs). However, it

xix

was detected production of auto-inducer two (AI-2) and presence of

diketopiperizines (DKPs) into the chemical extract obtained from TYEP medium

inoculated with P. fluorescens. The 16S rDNAs of strains 039, 059, 067, 068, 071,

and 099 isolated from raw milk were sequenced and they were identified as Pantoea

sp., Hafnia alvei, Enterobacter sp., Hafnia alvei, Hafnia alvei, and Aeromonas

hydrophila, respectively. It was verified that these strains presented different

spoilage potentials and resistance against different antibiotics. After cross-streak

assays in order to detect AHLs, only Pantoea sp. was not able to induce the monitor

strains. The thin layer chromatography and the chemical characterization of the

extracts by mass spectrometry confirmed that these strains produce different AHLs.

The quorum quenching mechanism was used and the lactonase enzyme was

expressed in the Enterobacter sp. transconjugant, which was unable to secrete AHLs

into LB medium. This strain was more proteolytic than the wild type, indicating that

the QS negatively regulates the proteolytic activity. Of the 32 restriction enzymes

used to digest the native plasmid from H. alvei 068 and 071, only DdeI, HinfI, MspI,

and RsaI were effective. Moreover, it was not possible to express lactonase in H.

alvei 068 and 071 transconjugants which compromised the evaluation of the

influence of the QS mechanism on spoilage activity. The halI gene, which encodes

the AHL synthase in H. alvei, was identified in the strains 059, 067, 068, and 071.

This gene was cloned, sequenced, and expressed in E. coli and it was verified that it

encodes a synthase responsible for the production of N-hexanoyl-DL-homoserine

lactone (C6-HSL) and N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL).

Besides producing AHLs, A. hydrophila produced chitinase, AI-2 and was

pathogenic against Caenorhabditis elegans.

INTRODUÇÃO GERAL

Em produtos lácteos refrigerados, a microbiota Gram-negativa é a mais

comumente encontrada, sendo o gênero Pseudomonas predominante no leite cru

refrigerado. Concentrações significativas de protease são encontradas no leite quando

a população de bactérias psicrotróficas está acima de 10

UFC/ml. Algumas dessas

bactérias secretam lipases que também estão envolvidas na deterioração dos produtos

lácteos. As enzimas proteolíticas e lipolíticas secretadas por Pseudomonas

fluorescens são codificadas pelos genes aprX e lipA, localizados em um mesmo

operon e cuja expressão é regulada por diferentes fatores.

A expressão gênica em muitas bactérias ocorre em resposta à densidade

populacional por um mecanismo chamado de quorum sensing. Este mecanismo

permite que as células controlem muitas de suas funções tais como, colonização de

superfície, motilidade, produção de exopolímeros, produção de antibióticos,

esporulação, formação de biofilme, bioluminescência, diferenciação celular,

competência para absorção de DNA, produção de pigmentos, transferência de

plasmídeos, esporulação, produção de toxinas, expressão de genes de virulência e

produção de enzimas hidrolíticas.

Quorum sensing é um mecanismo de comunicação entre células e é mediado

por sinais químicos extracelulares, denominados de moléculas auto-indutoras ou

sinalizadoras, produzidas pelas bactérias e liberadas no ambiente. Quando a

concentração do sinal é suficientemente alta, os genes alvo são ativados ou

reprimidos. A comunicação entre células pode ser bloqueada por meio da inativação

das moléculas sinalizadoras, pela interrupção dos genes que codificam enzimas que

sintetizam essas moléculas, ou pelo uso de aditivos que interfiram na ligação dessas

moléculas com a proteína receptora.

A utilização de culturas monitoras e de métodos de cromatografia líquida

acoplada a espectrometria de massa é fundamental para o conhecimento da estrutura

química das moléculas sinalizadoras e identificação de possíveis fenótipos regulados

pelo mecanismo de quorum sensing.

Moléculas sinalizadoras são produzidas por bactérias em alimentos e podem

estar associadas a processos de biodeterioração. O esclarecimento dos fatores

relacionados à regulação da atividade deterioradora de bactérias psicrotróficas torna-

se necessário considerando os diversos problemas tecnológicos e econômicos que

esse grupo de bactérias ocasiona à indústria de laticínios. Este estudo buscou a

purificação e caracterização de protease e lipase produzidas por P. fluorescens, bem

como, a caracterização das moléculas auto-indutoras do sistema de quorum sensing

em bactérias psicrotróficas como P. fluorescens, Pantoea sp., Hafnia alvei,

Enterobacter sp. e Aeromonas hydrophila isoladas de leite cru refrigerado e a

possível relação desse mecanismo com o processo de deterioração do leite.

Procedeu-se à redação dos capítulos desta tese, em língua inglesa, pelo fato

de a co-orientadora Dra. Anna Katharina Maria Riedel não dominar a língua

portuguesa.

CHAPTER 1

LITERATURE REVIEW

1.1. Psychrotrophic bacteria in milk

The microflora present in foods consists of microorganisms associated with

the raw material, microorganisms acquired during manipulation and processing, and

those which survived the stages of processing and storage of the product. Food

spoilage is a complex process and excessive amounts of foods are lost due to

microbial spoilage even when modern preservation techniques are employed (GRAM

et al., 2002). The establishment of contamination sources of foods is important in

order to control this process and to maintain the food microbial population the

smallest possible (JAY, 1996).

Milk constitutes an ideal medium for growth of deteriorative and pathogenic

microorganisms because of its high nutritional value, water content, and almost

neutral pH (FRANK, 1997). Bacteria can access milk and dairy products from

several sources, such as water, soil, animal feeding, milking equipments, and

manipulators (COUSIN, 1982; MUIR, 1996; ENEROTH et al., 1998; MURPHY and

BOOR, 2000).

Maintenance and transport of raw milk under refrigeration eliminate its

spoilage by mesophilic bacteria, but results in selection of psychrotrophic bacteria,

which are capable of multiply at 7 ºC or below, independent of their optimum growth

temperature (FRANK et al., 1992). In milk, psychrotrophs are mainly represented by

the gram-negative genus Pseudomonas, Achromobacter, Aeromonas, Hafnia,

Enterobacter, Serratia, Alcaligenes, Burkholderia, Chromobacterium and

Flavobacterium spp., and by the gram-positive genus Bacillus, Clostridium,

Corynebacterium, Streptococcus, Lactobacillus and Micrococcus spp (SØRHAUG

and STEPANIAK, 1997; PINTO 2004; MUNSCH-ALATOSSAVA and

ALATOSSAVA, 2006).

In refrigerated dairy products, gram-negative microflora is commonly found

(ADAMS et al., 1975; COUSIN, 1982; WIEDMANN et al., 2000; DOGAN and

BOOR, 2003). Pinto (2004) verified that gram-negative psychrotrophic proteolytic

bacteria constituted 84.6% of the isolates obtained from raw milk in Brazil, while the

proteolytic gram-positive bacteria constituted only 15.4%.

Psychrotrophic Pseudomonas predominate in refrigerated raw milk

(WIEDMANN et al., 2000; DOGAN and BOOR, 2003; PINTO, 2004), since they

present a well established physiologic mechanism of adaptation and growth at low

temperatures (JAY, 1996).

P. fluorescens constitutes the milk deteriorative species of higher importance

(WIEDMANN et al., 2000; DOGAN and BOOR, 2003; PINTO, 2004) due to its

ability to produce thermostable proteases that hydrolyze casein and decrease yield

and sensory quality of dairy products (SØRHAUG and STEPANIAK, 1997). Some

of these bacteria also secrete lecithinases and lipases that can play a significant role

in deterioration of these products (WIEDMANN et al., 2000; DOGAN and BOOR,

2003).

Among the microorganisms that survive pasteurization, sporeforming

psychrotrophs Bacillus spp. dominate (COUSIN, 1982). They secrete heat-resistent

extracellular proteases, lipases and phospholipase (lecithinase) that are of

comparable heat resistance to those of pseudomonads (SØRHAUG and

STEPANIAK, 1997).

1.2. Relevance of spoilage enzymes produced by psychrotrophic bacteria in milk

Spoilage of refrigerated raw milk occurs mainly due to protease and lipase

activities. Proteolytic enzymes produced by bacteria act on

-casein which results in

destabilization of casein micelles and gelation of milk (RECIO et al., 2000).

Lipolysis occurs due to the action of natural or microbial lipases. These enzymes are

able to hydrolise triglycerides, a milk fat constituent, in fatty acids of small chains

such as, butyric, caproic, caprylic and capric acid, mainly responsible for off-flavors

in milk and for rancidity in cheese (CHEN et al., 2003). Lipases produced by

psychrotrophs are more important than proteases in relation to development of

defects of flavor in cheese because proteases are soluble in water and lost in the

whey, while lipases are adsorbed in the fatty globules and retained in cheese mass

(FOX, 1989).

Proteases, lipases and phospholipases from psychrotrophic bacteria,

especially pseudomonads, are stable at high temperatures and survive pasteurization

and UHT treatment, but are not active above 50 to 60 °C (Sørhaug and Stepaniak,

1997). They show optima temperature at 30 – 45 °C, have low activation energy and

are therefore more active at 4 – 7 °C than enzymes from mesophilic microorganisms

(SØRHAUG and STEPANIAK, 1997).

The inactivation of 90% of extracellular activity of proteases produced by

Pseudomonas can be achieved at 72 °C for 4 – 5 h, or at 120 °C for 7 min (ADAMS

et al., 1975). However, these treatments are considered highly detrimental to milk

characteristics. Thermoresistant proteases produced by psychrotrophic bacteria from

Pseudomonas genus are alkaline metalloproteases, which need divalent ions as Ca

and Zn

for their stability and activity (RAO et al., 1998). Among the features that

stabilize thermoenzymes from psychrotrophic microorganisms are salt bridges,

additional hydrogen bonds, tighter Ca

-binding sites, maximized packing, shorter

loops, and an expanded hydrophobic core (SØRHAUG and STEPANIAK, 1997).

Psychrotrophic microorganisms or their enzymes in milk used for cheese

manufacture can result in cheese with various defects. In addition, in the cheese

industry, the producers have been problems of low yield due to bacterial protease

activity against casein (COUSIN, 1982). Development of off-flavours, including

bitterness and texture problems in cheese caused by proteases from psychrotrophs

have been reported, but only when psychrotroph counts in milk were 2 x 10

to 5 x

CFU/ml.

Even though in low concentration, proteases and lipases can cause defects in

products of long shelf-life such as UHT milk and milk powder (CELESTINO et al.,

1997). The fermentation rates of lactic acid bacteria during production of yogurt can

also be affected by growth of psychrotrophs in milk (FAIRBAIRN and LAW, 1986).

In some cases, the action of proteases produced by psychrotrophic bacteria results in

increased levels of peptides and free amino acids, which stimulate growth of lactic

acid bacteria (COUSIN and MARTH, 1977). On the other hand, lipolysis caused by

psychrotrophic bacteria increase the concentration of free fatty acids which may

inhibit the lactic acid bacteria (STOFER and HICKS, 1983).

1.3. Regulation of spoilage enzymes expression by P. fluorescens

Proteolytic and lipolytic enzymes secreted by P. fluorescens are codified by

aprX and lipA genes, respectively (BURGER et al., 2000; WOODS et al., 2001;

McCARTHY et al., 2004). These genes are located at opposite ends of an operon,

which also includes protease inhibitor (inh gene), type I secretion functions (aprDEF

genes) and two autotransporter proteins (prtA and prtB genes) (WOODS et al.,

2001).

Larger quantities of extracellular enzymes are produced at temperatures

below the optimum growth temperature. However, little is known about the

regulation mechanism involved in protease production at different temperatures.

According to Burger et al. (2000), the optimum growth temperature for

P. fluorescens LS107d2 is 27 ºC, and the optimum temperature for metalloprotease

production is between 22 and 27 ºC. However, above 27 ºC the proteolytic activity

decreased significantly. Production of metalloprotease above the optimum growth

temperature by P. fluorescens LS107d2 occurs in lower intensity and it is necessary

the production of PrtI and PrtR that are encoded by a dicistronic operon. Mutation in

prtI and prtR genes of P. fluorescens LS107d2 rendered cells unable to synthesize

protease above the optimum growth temperature. The PrtI activates transcription of

genes related to extracytoplasmatic functions, and protein PrtR, transmembrane, is an

activator of protein PrtI. When temperature increases, a conformational change

occurs in PrtR and this enzyme does not activate the PrtI. Then, PrtI does not activate

the transcription of aprX-lipA operon reducing the proteolytic activity. However, at

23 ºC, temperature below the optimum growth temperature of P. fluorescens

LS107d2, probably, another system acts controlling the production of protease, since

mutants as prtI

and prtR

showed proteolytic activity in this temperature.

In the soil bacterium, P. fluorescens M114, an iron-starvation

extracytoplasmatic function sigma factor, PbrA, required for transcription of

siderophore biosynthetic genes, was also implicated in protease regulation (SEXTON

et al., 1995; SEXTON et al., 1996). A serralysin-type metalloprotease gene, aprA,

was identified by Maunsell et al. (2006) and it was found to encode the major, if not

only, extracellular protease produced by this strain. The expression of aprA and its

protein product were found to be subject to complex regulation (MAUNSELL et al.,

2006). According to these authors, transcription analysis confirmed that PbrA was

required for full aprA transcription under low iron conditions, while the ferric uptake

regulator, Fur, was implicated in aprA repression under high iron conditions. Iron

regulation of AprA was dependent on culture conditions, with PbrA-independent

AprA-mediated proteolytic activity observed on skim milk agar supplemented with

yeast extract, when supplied with iron or purified pseudobactin M114. These effects

were not observed on skim milk agar without yeast extract. PbrA-independent aprA

expression was also observed from a truncated transcriptional fusion when grown in

sucrose asparagines tryptone broth supplied with iron or purified pseudobactin

M114. Thus, experimental evidence suggested that iron mediated its effects via

transcriptional activation by PbrA under low iron conditions, while an as-yet-

unidentified sigma factor(s) may be required for the PbrA-independent aprA

expression and AprA proteolytic activity induced by siderophore and iron.

Besides iron, the expression of protease and lipase in P. fluorescens B52 is

regulated by concentration of ions as Na

and K

and this involves homologue

proteins to the EnvZ-OmpR regulator system present in Escherichia coli and

Salmonella Typhimurium (McCARTHY, 2003). McKellar and Cholette (1986)

reported that in absence of ionic calcium an inactive precursor of protease was

produced by P. fluorescens. This precursor could not be activated proving that

calcium is required to stabilize the enzyme. Protease AprX production by

P. fluorescens CY091 was also dependent of CaCl

(LIAO and McCALLUS, 1998).

Rajmohan et al. (2002) verified that maximum production of protease by an isolate

of P. fluorescens was in minimal medium containing 1 mM/l of CaCl

. Therefore,

besides presence of calcium in the growth medium, carbon source causes a complex

effect in the synthesis of protease by Pseudomonas spp.

Protease production is induced by many products of protein degradation.

Asparagin appears to be the most effective inducer amino acid, whereas citric acid is

an inhibitor of the biosynthesis (McKELLAR, 1989). Glucose, galactose, lactate,

glutamine, and glutamic acid also delays or inhibits protease production by

P. fluorescens in raw milk (JASPE et al., 1994).

Moreover, production of protease and lipase by P. fluorescens and other

psychrotrophic bacteria in raw milk generally occur between the end of logarithmic

phase and beginning of stationary phase of growth (GRIFFITHS, 1989; MATSELIS

and ROUSSIS, 1998). Significant concentrations of metalloprotease (1.01 ng/ml)

were found into milk when population of P. fluorescens was above 10

CFU/ml

(BIRKELAND et al., 1985; MATTA et al., 1997, PINTO, 2004). Production of

extracellular enzymes at low cell density may not be advantageous to the cells due to

the distribution of enzymes and nutrients delivered into media (McCARTHY, 2003).

The expression of extracellular enzymes and metabolites by P. fluorescens is

generally regulated by GacS/GacA two-component system that occurs in a wide

range of gram-negative bacteria (BLUMER et al., 1999; KAY et al., 2005). The

small (119-nt) RNA, RsmX discovered by Kay et al. (2005), together with RsmY

and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-

binding proteins RsmA and RsmE and thereby, antagonize translational repression

exerted by these proteins in strain CHA0 (Figure 1) (KAY et al., 2005). This small

RNA triad was found to be both necessary and sufficient for posttranscriptional

derepression of biocontrol factors and for protection of cucumber from Pythium

ultimum. According to Kay et al. (2005), the same three small RNAs also positively

regulated swarming motility and synthesis of a quorum-sensing signal which is

unrelated to N-acylhomoserine lactones and which autoinduces the Gac/Rsm

cascade. Expression of RsmX and RsmY increased in parallel throughout cell

growth, whereas RsmZ was produced during the late growth phase. This differential

expression is assumed to facilitate fine tuning of GacS/A-controlled cell population

density-dependent regulation in P. fluorescens.

Figure 1 – Post-transcriptional control by Gac/Rsm signal transduction pathway in

P. fluorescens CHA0. The mRNAs that will be translated in proteins

responsible for protease activity (AprX), swarming motility, signal

molecules, and antibiotics are repressed by RsmA or RsmE. RsmX

together with RsmY and RsmZ forms a triad of GacA-dependent small

RNAs, which sequester the RNA-binding proteins RsmA and RsmE and

thereby antagonize translational repression exerted by these proteins

(source: http://www.unil.ch/dmf/page18042.html. Access on June 4

2007).

1.4. The mechanism of quorum sensing

Many bacteria regulate the expression of some genes in response to

population density in a mechanism known as quorum sensing (QS) (FUQUA et al.,

1994; WHITEHEAD et al., 2001). This mechanism allows cells to control many of

their functions such as surface colonization and motility, production of exopolymers,

production of antibiotics, biofilm development, bioluminescence, cell differentiation,

competence for DNA uptake, growth, pigment production, conjugal plasmid transfer,

sporulation, toxin production, virulence gene expression, and production of a range

of hydrolytic enzymes (SMITH et al., 2004).

Quorum sensing is a mechanism of cell-to-cell communication and it is

mediated by extracellular chemical signals denominated signal molecules or

autoinducers generated by bacteria when specific cell densities are reached. When

concentration of signal is sufficiently high, the target genes are either activated or

repressed. Bacteria have been reported to produce many different types of quorum

sensing signals. They can be grouped into amino acids and small peptides,

commonly used by gram-positive bacteria (MILLER and BASSLER, 2001;

WHITEHEAD et al., 2001) and fatty acid derivatives (N-acylhomoserine lactones)

or AHLs (autoinducer-1) frequently produced by gram-negative bacteria (FUQUA et

al., 1996; EBERL, 1999; WHITEHEAD et al., 2001). Other quorum sensing

signaling molecules have been identified in gram-negative bacteria: 2-heptyl-3-

hidroxy-4-quinolone (PQS) in Pseudomonas aeruginosa (PESCI et al., 1999;

EBERL, 2006) and diketopiperazines (DKPs) in P. aeruginosa and other bacteria

(HOLDEN et al., 1999; DEGRASSI et al., 2002). Recently, the autoinducer-2 (AI-2),

first discovered in Vibrio harveyi (BASSLER et al., 1993), has been described as a

new quorum sensing signal used by both gram-negative and gram-positive bacteria

(SCHAUDER and BASSLER, 2001; CHEN et al., 2002). Besides these signal

molecules, autoinducer-3 (AI-3) has been involved in QS mechanism, but it has

unknown chemical structure (SPERANDIO et al., 2003).

Other different chemical communication systems can also occur such as, in

Vibrio cholerae that posses, at least, three different QS systems that work in parallel

and regulate expression of virulence genes (MILLER et al., 2002). However, among

signaling molecules, AHLs, oligopeptides, and AI-2 are more known and used as

paradigma of QS (KELLER and SURETTE, 2006).

1.4.1. Biosynthesis and characterization of the signal molecules

1.4.1.1. Acyl-homoserine lactones (AHLs)

Frequently in gram-negative bacteria, AHLs are the signal molecules that are

diffused in the medium (EBERL, 1999). Chemical structure of AHLs indicates that

the acyl chain is derived from the metabolism of fatty acid, while molecules of

homoserine lactones originate from amino acids metabolism. Although all AHL

structures have the homoserine lactone ring moiety in common, the acyl side chain of

different AHLs can vary in length, degree of substitution, and saturation. The AHLs

currently identified have side chains that range from 4 to 18 carbons in length,

usually in increments of to 2-carbon units (ZHU at al., 2003). The overall length of

side chain and chemical modification at the β position provides specificity to QS

systems (FUQUA et al., 2001; ZHU et al., 2003).

Earliest described example of QS system using AHL is found in

bioluminescent marine symbiotic bacterium Vibrio fischeri. A protein known as LuxI

is responsible for production of AHL, and a protein called LuxR is an AHL receptor

as well as an AHL-dependent transcriptional activator of the luciferase operon

(FUQUA et al., 2001). Concentration of external AHL increases as a function of

population density of V. fischeri. When the AHL concentration reaches micromolar

range, the AHL can bind LuxR, and the resulting complex binds the promoter of lux

operon and activates its transcription.

LuxI-type proteins must carry out two reactions: formation of a homoserine

lactone ring from S-adenosylmethionine (SAM), and acylation of the amine at the

expense of acyl-ACP (HANZELKA and GREENBERG, 1995; JIANG et al., 1998;

SCHAEFER et al., 1996; VAL and CRONAN, 1998). This explains the variability of

these acyl groups, since ACPs can contain over 30 different acyl chains. The

homoserine lactone ring of AHL is unstable to hydrolysis, especially at alkaline pH.

AHLs are generally thought to diffuse readily across the bacterial envelope. Three

families of AHL synthases have been described. While most synthases resemble

LuxI of V. fischeri, two of these enzymes (AinS of V. fischeri and LuxM of V.

harveyi) resemble each other but do not resemble LuxI (GILSON et al., 1995).

However, both families of AHL synthases use the same substrates and may use

similar reaction mechanisms. A third AHL synthase, unrelated to either family, was

reported in P. fluorescens (LAUE et al., 2000).

1.4.1.2. Quinolones

Pesci et al. (1999) discovered that an additional signal, 2-heptyl-3-hydroxy-

4(1H)-quinolone, affects virulence gene expression in P. aeruginosa and more

detailed analyses revealed that this Pseudomonas quinolone signal (PQS) functions

as an integral component of QS network (DIGGLE et al., 2006). LasR regulates PQS

production, which, in turn, is necessary for transcription of the rhlR and rhlI genes,

thereby creating a regulatory link between las and rhl QS systems (PESCI et al.,

1999; McNIGHT et al., 2000). PQS is synthesized from anthranilate by head-to-head

condensation reaction between anthranilic acid and various β-keto fatty acids by the

products of the pqsABCDE operon (BREDENBRUCH et al., 2005). Although

additional work will be required to elucidate the complex interrelationships between

the two signaling pathways (EBERL, 2006), it is clear that PQS production is

modulated by both AHL-dependent QS systems and that exogenous addition of PQS

upregulates rhl-controlled QS phenotypes in P. aeruginosa (DIGGLE et al., 2003).

The pqsABCDE operon contains five genes that encode for a putative

coenzyme A ligase (pqsA), two β-keto-acyl carrier protein synthases (pqsB, pqsC),

and a FabH1 homologous transacetylase (pqsD). The pqsE gene appears to encode a

response effector protein which itself is not involved in biosynthesis of PQS.

Although the exact functions of the enzymes remain to be elucidated, it is clear that

pqsABCDE gene products direct the synthesis of 2-heptyl-4-quinolone (HHQ), the

immediate precursor of PQS (GALLAGHER et al., 2002). HHQ is thought to be an

extracellular messenger that is released from and taken up by P. aeruginosa cells

(EBERL, 2006). Once taken up, HHQ is converted into PQS by action of the putative

FAD-dependent non-oxygenase PqsH. Expression of the pqsH gene, which is not

physically linked to the pqsABCD operon, is partially controlled by the las system,

connecting AHL-dependent QS with PQS signaling (GALLAGHER et al., 2002).

Quinolone signaling was thought to be unique to P. aeruginosa, since PQS

molecules could not be detected in the culture supernatants of several other species

of Pseudomonas. However, Diggle et al. (2006) presented convincing evidence that

quinolone-dependent signaling is more widespread than so far anticipated. Diggle et

al. (2006) developed a simple and rapid method for screening bacterial culture

supernatants for AHQ production. To this end, a P. aeruginosa bioreporter was

constructed, which cannot synthesize AHQs due to the inactivation of the pqsA gene,

but which responds to exogenously supplied AHQs with the emission of light. This

biosensor strain can be incorporated within agar and used as an overlay following

thin layer chromatography (TLC) of the solvent-extracted culture supernatants.

According to Eberl (2006), using this approach as a fast initial screen and

liquid chromatography-mass spectrometry (LC-MS/MS) for confirmation of the

molecules identity, it was shown that several Burkholderia pseudomallei strains,

Burkholderia cenocepacia, Burkholderia thailandensis, and Pseudomonas putida

produce HHQ, but not PQS.

While in P. aeruginosa HHQ has to be converted to PQS before act as a

signaling molecule, in B. pseudomallei, HHQ serves as a signaling molecule per se

(DIGGLE et al., 2006). Considering that AHQs are synthesized from key cellular

metabolites, it appears likely that this class of signaling molecules is widely used by

bacteria (EBERL, 2006). With the AHQ biosensor development, an ideal tool is now

available to explore the full extent and diversity of AHQ-mediated signaling between

bacteria.

1.4.1.3. Diketopiperazines (DKP)

Although there is abundant literature data about dipeptides isolated from

microbial sources (HOLDEN et al., 1999; HOLDEN et al., 2000; BRELLES-

MARINÕ and BEDMAR, 2001; DEGRASSI et al., 2002; TAYLOR et al., 2004),

their true origin remains controversial. A variety of diketopiperazines (DKPs) were

shown to exist in protein hydrolysates as well as fermentation broths and cultures of

yeast, lichen, fungi, and bacteria (PRASAD, 1995). Microbial DKPs appear to be

both compounds synthesised de novo and catabolic products of peptone or other

components found in nutrient rich media (PRASAD, 1995; MITOVA et al., 2005).

Holden et al. (1999) found AHL-like molecules, DKPs, in supernatant of

many gram-negative bacteria, including P. fluorescens and P. aeruginosa as a

consequence of their ability to activate biosensors previously considered specific for

AHLs. Although DKPs are structurally quite distinct from AHLs, at high

concentrations they are able to cross-activate AHL-dependent reporter constructs

based on several different LuxR homologues (HOLDEN et al., 1999). Detection of

these DKPs appears to be an example of fortuitous chemical crosstalk and raises the

obvious question as to their origin and biological function(s) (HOLDEN et al., 2000).

Besides Pseudomonas, V. vulnificus also produces DKP, which was detected

by a QS bioindicator and it affected the expression of ToxR-dependent genes ompU

and ctxAB in Vibrio strains (PARK et al., 2006). Initially, it was assumed that this

pathogen would produce an AHL as do V. fisheri and V. harveyi, and various

bioindicators sensitive to AHL molecules were employed to detect QS signal

affecting expression of vvp and vvh in V. vulnificus. However, the active compound

from V. vulnificus which activated the QS bioindicator was a DKP molecule.

1.4.1.4. Furanosyl borate diester: auto-inducer two (AI-2)

While AHLs, PQS, and modified peptides (DKPs) described above are

confined to a reasonably narrow range of bacteria, recent evidence has suggested the

existence of a universal QS language (CÁMARA et al., 2002). A family of

molecules, termed AI-2, common to many gram-negative and gram-positive bacteria

has been described (FEDERLE and BASSLER, 2003). However, there is no direct

evidence for the involvement of AI-2 in regulation of pathogenic traits. Furthermore,

whether AI-2 has a true role in QS signaling in general has recently been questioned,

with suggestions that in most bacteria AI-2 is simply a metabolic side product, which

casts doubts on its suitability as a target in the context of QS inhibition.

Evidence for existence of AI-2 emerged with studies developed with

V. harveyi. Genetic evaluation of QS circuit in this bacterium showed that it produces

two auto-inducer signals in order to regulate bioluminescence. It seems that one

signal is used to intra-species communication and another to interspecies

communication (FEDERLE and BASSLER, 2003). In V. harveyi, the LuxI/R system

is absent and light production is controlled by two parallel, not homologue pathways

(BASSLER et al., 1993). In one of then, the auto-inducer molecule is not an AHL,

but a furanosyl borate diester (CHEN et al., 2002) which synthesis is controlled by

luxS, and the receptor protein is LuxQ, a kinase sensor of membrane (SURETTE et

al., 1999).

The occurrence of luxS-dependent AI-2 signaling is widespread among both

gram-negative and gram-positive bacteria. Moreover, 130 of 136 bacterial species

contain a highly conserved luxS homologue and a role for AI-2 in interspecies

communication has been proposed (SURETTE et al., 1999; BEESTON and

SURETTE, 2002).

The biosynthetic pathway for AI-2 synthesis was elucidated (SCHAUDER et

al., 2001; WINZER et al., 2002). In the cell, S-adenosylmethionine (SAM) is

consumed to form S-adenosylhomocysteine (SAH), which in turn, is hydrolysed by

the nucleosidase, Pfs, yielding adenine and S-ribosylhomocysteine (SRH).

Subsequently, the LuxS protein, a zinc metalloenzyme, converts

S-ribosylhomocysteine (SRH) to homocysteine and 4,5-dihydroxy-2,3-pentanedione

(DPD), a precursor probably requiring further rearrangement for AI-2 signal activity

(SCHAUDER et al., 2001; WINZER et al., 2002). Both luxS and pfs are required for

AI-2 activity. However, expression of luxS is constitutive while the transcription of

pfs is tightly correlated to AI-2 production and neither is regulated directly by AI-2

(BEESTON and SURETTE, 2002).

Although the role of AI-2 in V. harveyi as a density-dependent signal for

regulating bioluminescence has been well established, the function of AI-2 in other

bacteria has yet to be clarified. It is clear that AI-2 signaling regulates expression of

numerous genes and is involved in determining phenotypes, but exactly what AI-2 is

signaling is a murky subject. Emerging evidence indicates that AI-2 may not be a

density-dependent signal, but rather, a metabolic gauge or waste product (WINANS,

2002).

1.4.1.5. Oligopeptides

Opposite to gram-negative, gram-positive bacteria typically communicate by

using oligopeptide signals, which are synthesized as precursors on ribosomes and

proteolytically processed and released either by the general secretory pathway or by

a dedicated secretory apparatus (DUNNY and LEONARD, 1997). These signals are

generally detected using a two-component phosphorelay mechanism. In some cases,

the peptide receptor is a histidine kinase, while in other examples, the peptide is

imported by an ABC-type permease and the receptor is a cytoplasmic phosphor-

aspartate phosphatase (PEREGO, 1998). These signaling systems regulate processes

as pathogenesis, endospore formation, genetic competence, conjugation, and

production of microcin antimicrobials. Some gram-positive bacteria and at least, one

proteobacterium (Xanthomonas campestris) communicate via amphipathic

compounds called γ-butirolactones rather than peptides (BARBER et al., 1997).

1.4.2. Detection of signal molecules

AHL detection is based on different bacterial bio-assays. Steindler and

Venturi (2007) reviewed and discussed the currently available bacterial biosensors

which can be used in order to detect and study different AHLs molecules. All

bacteria reporter used have the genes that encode the AHL synthase inactivated by

mutation, but they have an AHL-responsive reporter gene. Expression of the reporter

gene is possible only in presence of exogenous AHLs. Reporter strains display

specificity towards different AHL molecules and the use of multiple reporters allow

detection of a wide range of AHLs and differentiation among many AHL production

patterns (Van HOUDT et al., 2004).

There are limitations when biosensors are used to detect AHL, and one must

be cautious in the interpretation of data obtained with AHL biosensors (STEINDLER

and VENTURI, 2007). Supernatant medium extracts might contain non-AHL

compounds that could potentially interfere or activate the biosensors response

(HOLDEN et al., 1999). In addition, from the negative response of AHL biosensors

one cannot rigorously conclude that the screened bacterial strain does not produce

AHLs. AHLs are usually active at very low concentrations and biosensor technology

allows for the quantification of AHLs. As different LuxR homologues have diverse

affinities with different AHLs, it is not accurate to compare the intensity of a

response of one AHL with the response obtained with a different AHL

(STEINDLER and VENTURI, 2007).

Methods involving liquid chromatography of reverse phase coupled with

mass spectrometry have been used to identify and to quantify AHLs that present

different chemistry structures (MIDDELETON et al., 2002).

Quorum-sensing molecule structures can be unequivocally assigned on the

basis of spectroscopic properties. Mass spectrometry (MS) detects even picomoles of

samples and can be coupled to gas chromatography (GC). Many types of ionization

are available, including electron impact (EI-MS), fast atom bombardment (FAB-MS)

and chemical ionization, and positive-ion atmospheric pressure chemical ionization

(APCI-MS). Analytical HPLC-mass spectrometry (LC-MS) is a very useful

technique that couples the resolving power of C18 reverse-phase HPLC directly with

mass spectrometry, such that the mass of the molecular ion (M+H)

and its major

component fragments can be determined for a compound with a given retention time.

This technique was used to determine the structure of different AHLs produced by

many bacteria.

1.4.3. Cross-communication

Signals released by one species might, under certain circumstances, be

detected by another species, either as an agonist or perhaps as antagonists. Synthesis

of proteases by Burkholderia cepacia was stimulated when cultured in spent medium

of P. aeruginosa; besides B. cepacia was shown to detect AHL released by P.

aeruginosa (McKENNY et al., 1995; RIEDEL et al., 2001). In some cases this could

be accidental rather than intentional. However, Salmonella and E. coli share a LuxR-

type protein called SdiA (MICHAEL et al., 2001), but do not encode a cognate AHL

synthase and do not release AHL. SdiA activates several genes in absence of AHL,

but only when overexpressed. Expression of SdiA at native levels induces, at least,

one gene in the presence of exogenous 3-oxo-octanoyl-HSL (MICHAEL et al., 2001).

Apparently, these bacteria, and possible others, have acquired an orphan receptor to

detect AHL produced by other bacteria species in mixed microbial communities.

Bruhn et al. (2004) demonstrated that H. alvei may induce food quality-

relevant phenotypes in other bacterial species in the same environment when H. alvei

718 and the S. proteamaculans B5a sprI mutant were coinoculated into milk. Milk

spoilage was observed when S. proteamaculans reach 10

CFU/ml and H. alvei

CFU/ml. No signs of spoilage were found at comparable cell densities when

coinoculating the S. proteamaculans B5a sprI mutant and H. alvei 718 halI mutant

into milk. Therefore, cross-communication is common among bacteria and it is

important to know which microorganisms produce these compounds in the

environment in order to control the QS mechanism.

1.4.4. Control of the quorum sensing system

The use of QS as a target to antimicrobial therapy can be done of different

ways. One of them consist in blocking microbial communication using auto-inducers

in order to prevent that bacteria express its virulence factors becoming, consequently,

harmless (KIEVIT and IGLEWSKI, 2000). This can be obtained by: (1) using

specific auto-inducers that bind to proteins like-LuxR but do not promote its

activation, and therefore, compromise these proteins to bind to the cognate auto-

inducer molecules. (2) Interrupting the biological reactions of syntheses of auto-

inducers using analogs of these compounds.

Intervention in bacterial mechanisms of QS can lead to discover new drugs

able to combat microorganisms resistant against known antibiotics (GIACOMETTI

et al., 2003). In addition to clinical relevance, interventions in QS system to control

microorganisms have applicability in agriculture. Auto-inducers produced by plants

can activate QS systems of pathogenic bacteria and stimulate early production of

virulence factors by bacteria, allowing the defense system of plants to recognize and

easily eliminates the infection (DONG et al., 2000). Another possibility to use QS in

agriculture consists in introduction of genes encoding enzymes evolved in

degradation of auto-inducers in plants, to protect them from infections caused by

pathogens as Erwinia caratovora (DONG et al., 2001).

Dong et al. (2000) discovered an enzyme in Bacillus sp. 240B1, known as

AiiA, which is able to degrade AHL. The aiiA gene encoding the lactonase from

Bacillus was cloned, sequenced, expressed in E. caratovora, and reduced production

of OHHL significantly. Also, it was observed a reduction of concentration of

pectinolitic enzymes in 10 folds, and decreased pathogenicity of this microorganism

against many plants (DONG et al., 2000). Lactonase enzyme hydrolyses lactone ring

of AHL molecules and it is present in many of Bacillus strains (LEE et al., 2002).

AiiA lactonase expression in B. thailandensis reduced accumulation of the signaling

molecules such as C6-HSL, C8-HSL, and C10-HSL and bacterial motility; increased

cellular generation time; and caused fluctuations in carbon metabolism (ULRICH,

2004). Heterologous expression system of Bacillus sp. lactonase in B. cepacia

species confirms AHL regulation in extracellular proteases production, swarming

motility, biofilm formation, and nematode pathogenicity (WOPPERER et al., 2006).

Another class of enzymes able to degrade AHL, known as acylase, was cloned,

expressed, and inhibited cell-to-cell communication in P. aeruginosa (LIN et al., 2003).

Another way to inhibit the gene expression mediated by AHL by interfering

with the ligation of the auto-inducer to LuxR (MANEFIELD et al., 2002) is

employing brominated furanones produced by Delisea pulchra (a microalga). The

interference of furanones with QS in bacteria was already showed (MANEFIELD et

al., 1999; RICE et al., 1999; REN et al., 2002). Besides furanones, Tateda et al.

(2001) verified that 2 μg/ml of azitromycin inhibited QS mechanism in P.

aeruginosa PAO1. They proposed that this antibiotic interferes with synthesis of

signaling molecules which leaded reduction of virulence factors production.

The screen for other compounds that inhibit QS is currently developed, and

Rasmussen et al. (2005) found that garlic extract was one of the most effective to

reduce P. aeruginosa biofilm tolerance to tobramycin treatment as well as virulence

in a Caenorhabditis elegans pathogenesis model.

1.4.5. Relevance of quorum sensing mechanism to the food industry

Although studies indicate that signaling compounds are produced by bacteria

in foods (GRAM et al., 1999; GRAM et al., 2002; CLOAK et al., 2002;

CHRISTENSEN et al., 2003; JAY et al., 2003; BRUHN et al., 2004; KASTBJERG

et al., 2007), the role of quorum sensing systems in foods is currently unknown.

Pinto et al. (2007) demonstrated that AHL-production is common among

proteolytic psychrotrophic bacteria isolated from raw milk. Once these organisms

were isolated from a common source, the possibility of cross-communication

between them is relevant and raises the question of what kind of phenotypes might

be regulated when they are growing together, and also, the relation of these

phenotypes with milk deterioration. The understanding of the role of QS mechanism

in regulation of spoilage phenotypes in bacteria from food origin is relevant and may

be used to create new ways to preserve food products (PINTO et al., 2007).

According to Pillai and Jesudhasan (2006), to understand the relationships

that exist between the food ingredients, food processing, handling, and food

consumption methods, and the mechanism of cell-cell communication based

microbial activity of pathogens and spoilage bacteria is very important.

Besides, the study of QS and its relationship with spoilage bacteria isolated

from foods is necessary in order to obtain a deep comprehension of the role of this

mechanism in food ecology. This knowledge may contribute for generation of new

strategies to control the growth of undesirable bacteria and the production of

detrimental metabolites, presenting a high potential for improving preservation and

safety of foods.

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CHAPTER 2

OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF

MILK-DETERIORATING HYDROLYTIC EXOENZYMES PRODUCED BY

Pseudomonas fluorescens

2.1. INTRODUCTION

The refrigeration of raw milk in the course of production has generally

improved its quality and shelf-life. However, this practice is responsible for new

quality problems due to the selection of psychrotrophic bacteria. Refrigeration does

not prevent the development of this microflora, which is able to produce heat-stable

extracellular enzymes such as proteases and lipases that subsequently degrade milk

components such as proteins and fat thereby reducing the shelf-life of milk and dairy

products (COUSIN, 1982; DECHEMI et al., 2005).

Many of these enzymes are produced by Pseudomonas fluorescens

(WIEDMANN et al., 2000; DOGAN and BOOR, 2003; McCARTHY et al., 2004).

As hydrolytic enzymes from P. fluorescens are not inactivated by pasteurization at

72 ºC for 15 s or by Ultra-High Temperature (UHT) treatment (GRIFFITHS et al.,

1981) they cause severe problems in the dairy industry such as, milk protein

hydrolysis, development of off-flavors, shelf-life reduction of dairy products,

decrease of yield during the cheese production, milk heat-stability loss, and gelation

of UHT milk (FAIRBAIRN and LAW, 1986; DATTA and DEETH, 2001; CHEN et

al., 2003).

Sørhaug and Stepaniak (1997) pointed out some important characteristics of

the metalloprotease secreted by P. fluorescens such as, optimum temperature

between 30 to 45 ºC, a significant residual activity at 4 ºC, and a pH optimum in a

neutral pH-range. The enzyme was described as metalloprotease, which contains one

zinc atom and up to eight calcium atoms conferring the thermostability to the protein.

Besides proteins, fats are important constituents of milk. The nutritional and

sensory value and the physical properties of a triglyceride are greatly influenced by

factors such as the position of the fatty acid in the glycerol backbone, the chain

length of the fatty acid, and its degree of unsaturation (SHARMA et al., 2001).

Microorganisms that produce lipolytic enzymes are important in the dairy industry

because they can produce rancid flavors and odors in milk and dairy products that

make these foods unacceptable to consumers (COUSIN, 1982). Lipase production by

P. fluorescens is influenced by the type and concentration of carbon and nitrogen

sources, iron, pH, dissolved oxygen concentration, and growth temperature

(COUSIN, 1982; BURGER et al., 2000; WOODS et al., 2001; RAJMOHAN et al.,

2002). Furthermore, McCarthy et al. (2004) demonstrated that lipase activity in the

culture supernatant of P. fluorescens B52 is regulated by the homologue of the

Escherichia coli EnvZ-OmpR two-component regulatory system.

The present work aimed to perform the molecular and biochemical

characterization of extracellular protease and lipase produced by the P. fluorescens

041. This strain was isolated from refrigerated raw milk, which was kept for 48 h at 4

°C in cooled tanks in a Brazilian farm. Both enzymes were overexpressed in

Escherichia coli, purified to heterogeneity by affinity chromatography and

biochemically characterized in order to evaluate their role in the degradation of milk

components.

2.2. MATERIAL AND METHODS

2.2.1. Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are listed in table 1.

Table 1 - Bacterial strains and plasmids used

Strain or plasmid Description Reference or source

Strains

E. coli XL1-Blue

Cloning and subcloning host

supE44, hsdR17, endA1, recA1, gyrA96, thi1, relA1,

lac- F´[proAB+, lacIq, lacZΔM15, Tn10 (tetF)]

Bullock et al., 1987

P. fluorescens 07A

and 041

Proteolytic psychrotrophic strains Martins et al., 2005

Plasmids

pCR2.1-TOPO

Cloning vector, lacZα fragment containing MCS, f1

origin, ColE1, Km

Invitrogen

pQE30-Xa

Vector for the insertion of a Factor Xa Protease

recognition site C-terminal of the 6xHis tag, T5

promoter,

lac operator, ribosome binding site, ATG start codon,

His tag sequence, multiple cloning sites, stop codons in

all three reading frames, Col E1 origin of replication,

Qiagen

pQE30-Xa-aprX041

1.43 kb fragment containing aprX from P. fluorescens

041 in pQE30-Xa, Ap

This study

pQE30-Xa-lipM041

1.42 kb fragment containing lipM from P. fluorescens

041 in pQE30-Xa, Ap

This study

2.2.2. Growth conditions

P. fluorescens 07A and 041 were cultured in TYEP (tryptone 1%, yeast extract

0.25%, KH

0.1%, K

HPO

0.1%, and CaCl

0.25%) broth at 25 °C with

aeration. Besides, these strains were inoculated into 12% (w/v) reconstituted skim

milk powder in order to verify their capacity to hydrolyze samples of milk after 18 h

at 25 °C. E. coli XL1-Blue was cultured in Luria-Bertani (LB) broth or on LB agar

plates at 37 °C, as required.

2.2.3. Protein quantifiction and enzyme assays

2.2.3.1. Protein quantification

The method of Bradford (BRADFORD, 1976) using bovine serum albumin as

a standard was used to quantify protein concentrations.

2.2.3.2. Protease assay

Proteolytic activity was determined as described before (CHRISTENSEN et

al., 2003). Briefly, this activity was investigated on azocasein by incubating 250 µl

of 2% azocasein (w/v) with 150 µl sterile filtered culture supernatant in TYEP or

with 75 µl of the purified AprX protease. The mixture was incubated at 30 °C for 12

h. Subsequently, the mixture was incubated at room temperature for 15 min with 1.2

ml of 10% (w/v) trichloroacetic acid (TCA), and centrifuged for 10 min at 15,000 g.

Prior to spectroscopic measurement, 600 µl supernatant were rescued and mixed with

750 µl 1M NaOH. The proteolytic activity was quantified by the determination of the

440

against a blank reaction mixture with 150 µl culture media or 75 µl Tris-HCl

20 mM, pH 8.0, CaCl

5 mM instead of the enzyme solution. Specific activity was

the unit of enzyme activity per hour per µg of protein.

2.2.3.3. Lipase assay

Lipolytic activity was determined as described before (CHRISTENSEN et al.,

2003). Briefly, this activity on p-nitrophenylpalmitate was investigated by incubating

1 ml of substrate (one volume 0.3% (w/v) p-nitrophenylpalmitate in isopropanol and

nine volumes 0.2% (w/v) sodium desoxycholate and 0.1% (w/v) gummi arabicum in

50 mM sodium phosphate buffer, pH 8.0) with 100 µl culture supernatant from

overnight cultures or with 50 µl of the purified lipase LipM for 20 min at room

temperature. Prior to spectroscopic measurement, 0.5 ml of 1 M Na

was added.

The lipolytic activity was quantified by the determination of the OD

410

against a

blank reaction mixture with 100 µl culture media or 50 µl Tris-HCl 20 mM, pH 8.0,

CaCl

5 mM instead of the enzyme solution. Specific activity was the unit of enzyme

activity per hour per µg of protein.

2.2.4. SDS-PAGE and zymograms

Proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE; LAEMMLI, 1970). After electrophoresis the gels were

stained with Coomassie brilliant blue.

Exoprotease activities of P. fluorescens culture supernatants, resolved

proteases after precipitation with ammonium sulfate, and recombinant expressed

AprX protease were visualized in SDS-PAGE-gels supplemented with 0.2% (w/v)

azocasein as described before (CHRISTENSEN et al., 2003). After electrophoresis,

proteins were renaturated by washing them twice in 50 mM Tris-HCl, pH 7.5, 25%

(v/v) isopropanol for 15 min at room temperature and once at 50 mM Tris-HCl, pH

7.5. After renaturation overnight at 4 °C in 50 mM Tris-HCl, pH 7.5, the zymogram

was incubated for 4 h in 5 mM CaCl

and 50 mM Tris-HCl, pH 8.0 at 40 °C. Prior to

detection, the gel was washed in 1 M NaOH for 5 min. Protease activity could be

detected as colourless zones in an orange background (CHRISTENSEN et al., 2003).

For the analysis of the lipase pattern after SDS-PAGE, proteins were

renaturated as described above. Finally, the gels were overlaid with the fluorescent

substrate methylumbelliferyl-butyrate (0.01 M in dimethylformamide) in order to

detect lipolytic activity using UV-light (360 nm) to visualize blue fluorescent bands

(RIEDEL et al., 2003).

2.2.5. Identification of proteins by mass spectrometry

P. fluorescens 041 and P. fluorescens 07A were grown in 1000 ml of TYEP

medium at 25 °C for 48 h. The cells were removed from the medium by

centrifugation at 10,000 g for 30 min, the supernatant was sterile filtered, and the

proteins were precipitated with ammonium sulfate (85% saturation). The samples

were centrifuged 20 min at 10,000 g and the supernatant was discarded. Then, the

pellets were washed twice with a 85% (w/v) ammonium sulfate and centrifuged

again. The pellets were dissolved in 50 mM Tris-HCl, pH 8.0 and dialyzed overnight

at 4 °C against 50 mM Tris-HCl, pH 8.0, 5 mM CaCl

. Aliquots of 15 µl of the

dialysed samples were loaded on the SDS-PAGE (12%) and Coomassie-stained

protein bands were excised, digested with trypsin and analysed by mass spectrometry

as described before (RIEDEL et al., 2005). Briefly, gel pieces stained were treated

with 50 µl of destaining solution (50% methanol in 100 mM NH

HCO

, pH 8.0) for

30 min at 37 °C. The gel pieces were washed twice with 100 µl of water and once

with 100 µl of 100 mM NH

HCO

buffer. This buffer was removed and the gel

pieces were dehydrated in 100 µl of 80% (v/v) acetonitrile in 20% water for 10 min.

After solvent removal, the residual solvent was evaporated for 30 min at 50 °C.

Aliquots of 10 µl of trypsin solution (20 ng of trypsin in 2 ml of 5 mM Tris buffer,

pH 8.4) were incubated 15 min with the gel pieces at room temperature. Finally, 10

µl of 5 mM Tris buffer, pH 8.4 were added and the samples were incubated for at

least 3 h at 37 °C. The samples were kept at -20 °C until they were analysed by mass

spectrometry.

The samples were analyzed on a 4700 Proteomics Analyzer MALDI

TOF/TOF system (Applied Biosystems, Framingham, MA). The instrument was

equipped with an Nd:YAG laser operating at 200 Hz. All mass spectra were recorded

in positive reflector mode, and were generated by accumulating data from 5,000 laser

pulses. First, MS spectra were recorded from the standard peptides on each of the six

calibration spots, and the default calibration parameters of the instrument were

updated. Subsequently, MS spectra were recorded for all sample spots on the plate

and internally calibrated using signals from autoproteolytic fragments of trypsin. Up

to five spectral peaks per spot that met the threshold criteria were included in the

acquisition list for the MS/MS spectra. Peptide fragmentation was performed at

collision energy of 1 kV and a collision gas pressure of approximately 2.561027

Torr. During MS/MS data acquisition, a method with a stop condition was used. In

this method, a minimum of 3000 laser pulses and a maximum of 6000 laser pulses

were allowed for each spectrum.

For the protein identification, MS data were searched using MASCOT

version 1.9.05 (Matrix Science, London, UK) as the search engine. All searches were

performed against a database comprising annotated proteins. GPS (Global

Proteomics Server) Explorer Software (Applied Biosystems) was used for submitting

data acquired with the MALDI-TOF/TOF mass spectrometer for database searching.

The following search settings were used: maximum number of missed cleavages: 1;

peptide tolerance: 25 ppm; MS/MS tolerance: 0.2 kDa. Carboxyamidomethylation of

cysteine was set as fixed modification, and oxidation of methionine was selected as

variable modification.

2.2.6. DNA manipulation, PCR reaction and sequencing

2.2.6.1. DNA manipulations

Cloning, restriction enzyme analysis, and transformation of E. coli were

performed essentially as described previously (SAMBROOK et al., 1989). PCR was

performed with TaKaRa Ex Taq polymerase (TaKaRa Shuzo, Shiga, Japan). Plasmid

DNA was isolated using the QIAprep Spin Miniprep kit, and chromosomal DNA was

purified with the DNeasy tissue kit. DNA fragments were purified from agarose gels

by using the QIAquick gel extraction kit (all kits from Qiagen, Hilden, Germany).

2.2.6.2. Amplification of the protease and lipase genes by PCR

The PCR reaction consisted of 2.0 mM MgCl

, 5.0 µl of 10X buffer Ex Taq,

2.5 mM deoxynucleotide triphosphates (dNTPs), 25 pmol of each primer, 1 U Ex

Taq DNA polymerase, and 40 ng of DNA in a final volume of 50 µl. Primers based

on the sequences of the aprX (GenBank accession numbers DQ146945, AY298902,

AF216700) and lip gene (GenBank accession numbers AF216702, AY694785,

M86350, S77830, D11455, AB063391, AY304500, AY673674) of other P.

fluorescens strains were designed (Table 2), and synthesized by Microsynth (Zürich,

Switzerland). PCR-reactions were carried out in a T3 thermocycler (Biometra

Biolabo Scientific Instruments, Zürich, Switzerland).

Table 2 - Primers used to amplify the aprX and lipM genes by PCR

Primer Sequence (5’-3’) Aplification

Apr-F TTATGTCAAAAGTAAAAGAC Amplification of the aprX gene

Apr-R TCAGGCTACGATGTCACTG Amplification of the aprX gene

APRX-F ATTGGATCC

AAAGCTATTGTATCTGCCGCG Amplification of the aprX gene

and preparation for cloning in

pQE-30Xa

APRX-R ATTGAGCTC

TCAGGCTACGATGTCACTGGC Amplification of the aprX gene

and preparation for cloning in

pQE-30Xa

Lip-F ATGGGTRTSTTYGACTATAAAAACC Amplification of the lipM gene

Lip-R TTAACCGATCACAATCCCCTCC Amplification of the lipM gene

LIPM-F ATTGGATCC

AACCTCGGTACCGAGGACTC Amplification of the lipM gene

and preparation for cloning in

pQE-30Xa

LIPM-R ATTGAGCTC

TTAACCGATCACAATCCCCTCCC Amplification of the lipM gene

and preparation for cloning in

pQE-30Xa

The introduced restriction sites BamHI and SacI are underlined.

2.2.6.3. Sequencing of the protease and lipase genes

The M13 Forward and Reverse primers were used to sequence the aprX and

lipM genes of P. fluorescens 07A and 041 cloned into pCR2.1-TOPO.

2.2.7. Cloning, heterologous expression and purification of P. fluorescens 041

protease and lipase

Once the complete sequences of the aprX and lipM genes were obtained,

primers (Table 2) were designed to amplify the open reading frame (ORF) by PCR

using the bacterial genomic DNA as a template and TaKaRa Ex Taq as DNA-

polymerase. The primers generated BamHI and SacI sites at the 5’ and 3’ ends of the

amplificates, respectively.

The DNA amplificates, 1434 bp and 1422 bp, containing the aprX and lipM

structural genes respectively were digested with BamHI and SacI and ligated into the

vector pQE-30Xa (Qiagen), which contains 6xHis-tag coding sequence either 5' or 3'

to the cloning region, cut with the same restriction enzymes. Plasmids harbouring the

ORF of aprX or lipM inserted downstream of the T5 promoter and two lac operator

sequences which increase lac repressor binding and ensure efficient repression of the

powerful T5 promoter were selected and named pQE-30Xa-aprX041 or pQE-30Xa-

lipM041, respectively. The plasmids were subsequently transformed into expression

strain E. coli XL1-Blue.

For overproduction of AprX and LipM, E. coli XL1-Blue cells carrying pQE-

30Xa-aprX041 or pQE-30Xa-lipM041 were grown in dYT medium (tryptone 1.6%,

yeast extract 1.0%, NaCl 0.5%, and glucose 0.2%) containing ampicillin (100 µg

-1

) at 37 °C under vigorous shaking. At an optical density of 0.5 at 600 nm,

isopropyl-β-D-thiogalactopyranoside (IPTG) which binds to the lac repressor protein

and inactivates it was added to the culture to a final concentration of 1 mM in order

to induce the expression of aprX and lipM.

After 5 h incubation at 37 °C, the cells were collected by centrifugation at

10,000 g for 30 min, resuspended in 50 mM Tris-HCl (pH 8.0) and centrifuged at

10,000 g for 30 min followed by two washing steps with 50 mM Tris-HCl pH 8.0,

NaCl 150 mM. The resulting cell pellets were finally resuspended in lysis buffer (8

M urea, 0.1 M NaH

, 0.01 M Tris-HCl, pH 8.0) and the recombinant enzymes

were purified under denaturing conditions using the Ni-NTA Spin Columns (Qiagen)

according to the suppliers’ instructions. After purification, the enzymes were

subjected to dialysis (20 mM Tris-HCl, pH 8.0, 5 mM CaCl

) overnight at 4 °C to

allow renaturation of the enzymes AprX and LipM.

2.2.8. Biochemical characterization of purified enzymes

2.2.8.1. Temperature optimum

Proteolytic and lipolytic activities of purified AprX and LipM were

determined as described above on azocasein and p-nitrophenyl palmitate,

respectively, at various incubation temperatures (4, 25, 30, 37, 40, 45, 50, and 60 ºC).

2.2.8.2. pH optimum

Proteolytic and lipolytic activities of purified AprX and LipM were

determined as described above on azocasein and p-nitrophenyl palmitate,

respectively, at various pH. The following buffer systems in a final concentration of

50 mM were used: sodium succinate (pH 4.0, 5.0, 6.0), Tris-HCl (pH 6.0, 6.5, 7.0,

7.5, 8.0, 8.5, 9.0), and glycine-NaOH (pH 9.0, 10.0, 11.0, 12.0, 13.0).

2.2.8.3. Heat stability

Purified enzymes AprX and LipM were incubated for 5, 10, 15, 20, 30 and 60

min. at 50, 60, 70, 80, 90, and 100 °C to determine their thermostability. Moreover,

they were incubated at 65 °C for 30 min. and 72 °C for 20 s to simulate the milk

pasteurization treatments. The final activity was determined as described above.

2.2.8.4. Metal ions

To investigate the effect of metal ions on purified AprX and LipM, the

reaction mixture was supplemented with 1 mM of each compound (MnS0

, CoCl

ZnSO

, FeSO

, MgSO

, or FeCl

) as described by Setyorini et al. (2006). Proteolytic

and lipolytic activities were determined on azocasein or p-nitrophenyl palmitate as

described above.

2.2.8.5. Protease inhibitors

The effect of potential protease inhibitor on the proteolytic activity of purified

AprX was determined as described by Setyorini et al. (2006) by supplementing the

reaction mixture with 1 mM PMSF, 1 mM EDTA, 1 mM Pefabloc SC, 2% (w/v)

SDS, 4 M urea, 0.1% (w/v) DTT, and 0.1% (v/v) β-mercaptoethanol and subsequent

measurement of residual activities on azocasein as described above.

2.2.9. Substrate specificity

2.2.9.1. Protease

The substrate specificity of purified AprX was determined on casein, elastin,

collagen, bovine serum albumin, and gelatine. The reaction mixture consisted of

0.4% (w/v) of each protein in 400 µl of 50 mM Tris-HCl, pH 6.5 and 150 µl of

enzyme solution. After incubation at 37 °C for 1 h, the mixture was withdrawn and

the increase in the amount of free amino groups was determined by the ninhydrin

method according to Setyorini et al. (2006).

2.2.9.2. Lipase

Activities of purified LipM on different p-nitrophenyl fatty acid esters (p-

nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl palmitate, and p-

nitrophenyl phosphorylcholine) were measured according to the assay for lipolytic

activity as described above.

2.3. RESULTS AND DISCUSSION

2.3.1. Milk-deteriorating hydrolytic activities of P. fluorescens

P. fluorescens 041 showed higher proteolytic (Figure 1A) and lipolytic

(Figure 1B) activities in the supernatant of TYEP medium than the strain 07A.

Moreover, strain 041 exhibited a higher capacity to hydrolyse milk than P.

fluorescens 07A when both strains were inoculated into 12% (w/v) reconstituted

skim milk powder (Figure 1C). SDS-PAGE analysis of ammonium sulfate

precipitated protein from supernatants of TYEP cultures of P. fluorescens 07A and

041 demonstrated the presence of multiple protein bands (Figure 2, lines 1 and 3).

Proteolytic activity of the dominant 50 kDa band was demonstrated by a zymogram

incorporating azocasein (Figure 2, lines 2 and 4). Mass spectrometry analysis of the

major proteolytic protein band identified this protein as metalloprotease, which was

designated as AprX. Previously, numerous Pseudomonas spp. have been shown to

produce and secrete hydrolytic enzymes (LIAO and MCCALLUS, 1998; BURGER

et al., 2000; WOODS et al., 2001; McCARTHY et al., 2004; MAUNSELL et al.,

2006). Rajmohan et al. (2002) verified the production of five proteases by a single

strain of P. fluorescens. However, in this work, mass spectrometry analysis of low

molecular bands that showed also proteolytic activity (Figure 2) revealed that these

bands were degradation products of AprX.

Surprisingly, no lipolytic activity could be detected when the renaturated

SDS-PAGE was overlayed with the lipase substrate methylumbeliferyl-butyrate.

Probably it occurred due to the degradation of lipase by protease or because Ca

was

not added into the renaturation buffer and LipM needed this ion for correct folding.

Figure 1 – Production of extracellular hydrolytic enzymes by P. fluorescens. A:

Proteolytic activity in the supernatant of TYEP medium; B: Lipolytic

activity in the supernatant of TYEP medium; C: Samples of

reconstituted skin milk powder (12%) inoculated with P. fluorescens

07A and 041 after 18 h of incubation at 25 °C. Data represent the

average of duplicate experiments.

0.000

0.005

0.010

0.015

0.020

0.025

0.030

07A O41

P. fluorescens

Proteolytic activity (Units h

-1

µg

-1

)

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

07A O41

P. fluorescens

Lipolytic activity (Units h

-1

µg

-1

)

07A

041

A B

Figure 2 – Coomassie-stained SDS-PAGE and azocasein zymogram on 12% sodium

dodecyl sulfate-polyacrylamide gels visualizing protease production by P.

fluorescens after grown in TYEP medium supplemented with 0.25%

CaCl

. Lane S: molar mass standards (BioRad); lane 1: SDS-PAGE of

ammonium sulfate precipitated proteins of P. fluorescens 07A

supernatant; lane 2: azocasein zymogram of ammonium sulfate

precipitated proteins of P. fluorescens 07A supernatant; lane 3: SDS-

PAGE of ammonium sulfate precipitated proteins of P. fluorescens 041

supernatant; lane 4: azocasein zymogram of ammonium sulfate

precipitated proteins of P. fluorescens 041 supernatant.

2.3.2. Cloning and sequencing of protease and lipase genes

Primers based on sequences of protease and lipase from other P. fluorescens

strains were synthesized and used to amplify a segment encoding these enzymes in

P. fluorescens 041. Electrophoresis of the PCR products revealed a single product at

about 1,500 bp for both enzymes.

Subsequently, the protease and lipase genes were sequenced. The aprX and

the lipM genes of P. fluorescens 041 comprised 1,434 and 1,425 bp, respectively,

and coded for proteins with 477 and 474 amino acids. Based on amino acid

sequence, the the molecular mass of both enzymes was predicted to be 49.365 kDa

and 49.811 kDa, which could be confirmed by SDS-PAGE analysis of purified

250

150

100

S 1 2 3 4

enzymes (Figure 3, lane 3 and 5). These results were similar to those found for

protease and lipase of Pseudomonas strains isolated from raw milk (MAKHZOUM

et al., 1996; KIM et al., 1997; LIAO and MCCALLUS, 1998; RAJMOHAN et al.,

2002; KOJIMA and SHIMIZU, 2003).

The aprX gene of P. fluorescens 07A and P. fluorescens 041 presented 96%

identity with each other. When the sequence of aprX gene of P. fluorescens 041 was

compared to the sequences of the GenBank, it was verified 97% identity with both

extracellular alkaline metalloprotease (aprX) gene of P. fluorescens strain A506

(accession number

AY298902) and with the protease (aprX) gene of P. fluorescens

strain F (accession number

DQ146945). On the other hand, the lipM gene of P.

fluorescens 07A and P. fluorescens 041 showed 94% identity with each other, and

this gene of P. fluorescens 041 showed 93% identity with polyurethanase lipase A

(pulA) gene of P. fluorescens (accession number AF144089) and 86% with the lipase

(lipA) gene of P. fluorescens (accession number AF216702).

Once the AprX and LipM produced by strains of P. fluorescens isolated from

raw milk showed high identity with the sequences from homologous enzymes in the

data base, it will be possible to use the aprX and lipM genes as markers to detect

spoilage psychrotrophic bacteria in milk using the PCR technique as described by

Martins et al. (2005). According to these authors, the technique could reduce the time

for detection of these bacteria in raw milk allowing processor to decide about the

best use of milk during processing. Besides, the characterization of these spoilage

enzymes is important to estimate the degradation of milk components, and thus

further improve enzymatic methods to access the quality of milk.

2.3.3. Overexpression and purification of AprX and LipM

The expression and purification of recombinant proteins facilitate production

and detailed characterization of proteins. After cloning of alkaline metalloprotease

AprX and lipase LipM in the vector pQE30Xa and heterologous expression in E. coli

XL1-Blue (Figure 3, lane 1 and 2), the two proteins were purified under denaturing

conditions as the purification of overexpressed AprX and LipM under native

conditions was hampered by massive occurrence of inclusion bodies. Purified

proteins were renatured by dialysis against 20 mM Tris-HCl, pH 8.0, 5 mM CaCl

The AprX and LipM were purified to homogeneity (Figure 3, lane 3 and 5),

showed the expected molecular mass and were active on zymograms after

renaturation when 1 mM of CaCl

was added into the renaturation buffer (Figure 3,

lane 4 and 6).

Figure 3 - Coomassie-stained SDS-PAGE and zymogram on 12% sodium dodecyl

sulfate-polyacrylamide gels visualizing recombinant AprX and LipM.

Lane S: molar mass standard (BioRad); lane 1: SDS-PAGE of crude

extract of E. coli XL1-Bue carrying pQE30-Xa-aprX-041; lane 2: SDS-

PAGE of crude extract of E. coli XL1-Bue carrying pQE30-Xa-lipM-041;

lane 3: SDS-PAGE of purified AprX; lane 4: azocasein zymogram of

purified AprX; lane 5: SDS-PAGE of purified LipM; lane 6:

methylumbelliferyl-butyrate zymogram of purified LipM.

2.3.4. Biochemical characterization of AprX and LipM

The temperature optimum of the purified protease of P. fluorescens 041 was

37 °C (Figure 4). This is in agreement to the temperature optimum of the most other

pseudomonal proteases, which are between 30 and 45 °C (FAIRBAIRN and LAW,

S 1 2 3 4 5 6

250

150

100

1986; SØRHAUG and STEPANIAK, 1997). Besides, the protease showed

considerable activity under conditions of refrigeration from 4 °C to 7 °C, and low

activity in temperatures higher than 45 °C (Figure 4). As this enzyme has a low

activation energy and is therefore more active at 4 °C to 7 °C than enzymes from

mesophilic microorganisms (SØRHAUG e STEPANIAK, 1997), it is readily able to

degrade casein and cause many problems to the dairy industry.

Figure 4 – Effect of temperature on activity of purified AprX of P. fluorescens 041

on azocasein. Data represent the average of duplicate experiments.

The temperature optimum of the purified lipase was 25 °C (Figure 5).

Different from AprX, LipM showed only a residual activity of 3.7% at 4 °C, and

exhibited low activities at temperatures higher than 37 °C (Figure 5). These data

were different from thouse obtained by Makhzoum et al. (1996) who detected

temperature optimum of a purified lipase from P. fluorescens 2D as 40 °C.

Temperature optimum from 20 until 35 °C has also been reported for lipases from P.

fluorescens (DRING and FOX, 1983; ROUSSIS et al., 1998).

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0 5 10 15 20 25 30 35 40 45 50 55 60

Temperature (°C)

Proteolytic activity (Units h

-1

µg

-1

)

Figure 5 - Effect of temperature on activity of purified LipM of P. fluorescens 041

on p-nitrophenyl palmitate. Data represent the average of duplicate

experiments.

The effect of pH of the assay conditions on extracellular protease activity is

shown in Figure 6. It had a broad pH range of activity towards azocasein as substrate.

The pH optimum of AprX is at 6.0 to 6.5 (Figure 6), which is close to the pH of

bovine milk (6.66). Sørhaug and Stepaniak (1997) also pointed out the pH optimum

of this enzyme close to neutrality. Besides, the protease exhibits still 36% residual

activity at pH 4.0 and 62% at pH 9.0.

On the other hand, LipM showed a narrow range of activity with the highest

at pH 7.5 (Figure 7). At pH values lower than 6.0 and higher than 11.0 only residual

lipase activities could be detected (Figure 7). Range of pH optimum from 7.0 to 9.0

has been reported for lipases from P. fluorescens (SZTAJER et al., 1991;

MAKHZOUM et al., 1996; ROUSSIS et al., 1998).

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0 5 10 15 20 25 30 35 40 45 50 55 60

Temperature (°C)

Lipolytic activity (Units h

-1

µg

-1

)

Figure 6 – Effect of pH on proteolytic activity of purified AprX of P. fluorescens 041

on azocasein. Data represent the average of duplicate experiments. (▲)

Succinate buffer, (■) Tris/HCl buffer, and (●) NaOH-glycine buffer.

Figure 7 – Effect of pH on lipolytic activity of purified LipM of P. fluorescens 041

on p-nitrophenyl palmitate. Data represent the average of duplicate

experiments. (▲) Succinate buffer, (■) Tris/HCl buffer, and (●) NaOH-

glycine buffer.

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

Proteolytic activity (Units h

-1

µg

-1

)

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Lipolytic activity (Units h

-1

µg

-1

)

The protease activity was strongly decreased by temperatures of 50, 60, 70,

80, 90 and 100 °C maintaining the residual activity between 2 and 4% after 60 min

(Figure 8). The inactivation of 90% of activity of extracellular protease produced by

Pseudomonas could be reached at 72 °C for 4 to 5 h or at 120 °C for 7 min (ADAMS

et al., 1975).

Figure 8 – Effect of heat treatment for 60 min on proteolytic activity of purified

AprX of P. fluorescens 041. Data represent the average of duplicate

experiments.

However, these treatments are considered highly prejudicial to the milk

characteristics. Inactivation of the metalloprotease at temperature and time

conditions used during the pasteurization process was also evaluated. AprX showed

70% residual activity when it was treated at 75 °C for 20 s (HTST treatment - high

temperature and short time) and 4% residual activity when it was incubated at 65 °C

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

Proteolytic activity (Units h

-1

µg

-1

)

X 37 °C

○ 50 °C

□ 60 °C

Δ 70 °C

● 80 °C

◊ 90 °C

■ 100 °C

for 30 min (LTLT - low temperature and long time). This demonstrates the

importance of the combination of time and temperature during the heat treatment

once, when the time was increased the activity decreased significantly. As the heat

treatment and the refrigeration temperature adopted during the storage of milk

neither complet inhibit the activity of this spoilage enzyme nor the growth of

psychrotrophs, it is necessary to adopt the good manufactory practices (GMP) to

limit the contamination of raw milk.

Besides protease, the inactivation of lipase by heat has been important to

dairy processors because enzymes that survive pasteurization can be detrimental to

keeping quality of products. The residual activity of the lipase after 60 min pre-

incubation at 50, 60, 70, 80, 90 and 100°C was between 0 and 6%. It was observed a

little residual activity after all thermal treatment, but the residual activity was

abolished after the treatment of the lipase at 100 °C for 30 min (Figure 9).

Figure 9 – Effect of heat treatment for 60 min on lipolytic activity of purified LipM

of P. fluorescens 041. Data represent the average of duplicate

experiments.

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

Lipolytic activity (Units h

-1

µg

-1

)

x 25 °C

○ 50 °C

□ 60 °C

Δ 70 °C

● 80 °C

◊ 90 °C

■ 100 °C

The treatment of 65 °C for 30 min (LTLT), and 75 °C for 20 s (HTST)

similar to pasteurization conditions reduced the lipolytic activity to 13.2% and

25.4%, respectively. According to Cousin (1982), complete inactivation of lipases

was obtained by autoclaving milk at 121 °C for 15 min. Knaut (1978) observed that

lipases from P. fluorescens species were stable above 100 °C, and a heat-treatment of

98 °C for 14 to 25 min was necessary to inactivate the lipases of some Pseudomonas

species, including P. fluorescens and P. fragi.

The correct protein folding of AprX seems strongly dependent of Ca

as no

renaturation of AprX could be achieved when the purified enzyme solution was

dialyzed against buffer lacking this metal ion. Surprisingly, some metal ions e.g.

even reduced the proteolytic activity significantly (Table 3).

Like protease, the maximum activity of the lipase was observed when Ca

was added in the dialyse solution. On the other hand, 1 mM of the other ions strongly

reduced the lipolytic activity (Table 3). According to Makhzoum et al. (1996),

calcium had a strong activating effect on the lipase (125% increase in activity),

suggesting the possible effect of this cation on the activity and stability of the

enzyme.

Table 3 - Effects of metal ions on the activities of alkaline metalloprotease and lipase

Relative activity (%)

Metal ion

Alkaline metalloprotease

Lipase

None 100 100

73 61

48 59

86 49

90 48

102 65

100 50

A reaction mixture containing 250 µl of 2% (w/v) azocasein in 50 mM Tris/HCl (pH 8.0), 75 µl of

AprX, and 1 mM of each metal ion was incubated at 37 °C for 12 h. The remaining activity was then

measured, as described in the text.

A reaction mixture containing 1 ml of substrate (one volume of 0.3% (w/v) p-nitrophenyl palmitate in

isopropanol and nine volumes 0.2% (w/v) sodium desoxycholate and 0.1% (w/v) gummi arabicum in

50 mM sodium phosphate buffer, pH 8.0), 50 µl of LipM, and 1 mM of each metal ions was incubated

at 25 °C for 30 min. The remaining activity was then measured, as described in the text.

The effect of different protease inhibitors on AprX activity is shown in

Table 4. The activity was strongly decreased when 1 mM of EDTA, an inhibitor that

specifically inhibits metalloproteases was added to the reaction mixture confirming

the metalloprotease nature of the enzyme. In addition, AprX was strongly inhibited

by denaturing and reducing agents such as SDS, dithiothreitol (DTT), β-

mercaptoethanol, and urea (Table 4).

Table 4 - Effects of inhibitors and denaturing and reducing agents on the activity of

alkaline metalloprotease

Compound Relative activity (%)

Inhibitor

None 100

PMSF 95

EDTA 51

Pefabloc SC 89

Denaturing and reducing agent

None 100

SDS 6

Urea 38

DTT 24

β-mercaptoethanol 44

A reaction mixture containing 250 µl of 2% (w/v) azocasein in 50 mM Tris/HCl (pH 8.0), 75 µl of

AprX, and 1 mM of each inhibitor was incubated at 37 °C for 12 h. The remaining activity was then

measured, as described in the text.

A reaction mixture containing 250 µl of 2% (w/v) azocasein in 50 mM Tris/HCl (pH 8.0), 75 µl of

AprX, and 2% (w/v) SDS, 4 M urea, 0.1% (w/v) DTT, or 0.1% (v/v) β-mercaptoethanol in 50 mM

Tris/HCl (pH 8.0) was incubated at 37 °C for 12 h. The remaining activity was then measured, as

described in the text.

The alkaline metalloprotease was furthermore tested for its capability to

hydrolyse different substrates. Highest activity was found on gelatine (100%) and

casein (87.6%), followed by collagen (57%), elastin (41.2%), and bovine serum

albumin (39.8%).

LipM was also tested for its capability to hydrolyse different substrates.

Highest activity was found on p-nitrophenyl palmitate (100%), followed by p-

nitrophenyl butyrate (73%), p-nitrophenyl acetate (20%), and p-nitrophenyl

phosphorylcholine (11%). These results confirmed that this enzyme has lipolytic

activity rather than esterase activity.

2.4. CONCLUSIONS

The aprX and the lipM genes are highly conserved among P. fluorescens

strains and encode protease (AprX) and lipase (LipM), which have molecular mass

of 50 kDa and are dependent of Ca

Metalloprotease from P. fluorescens 041 exhibits temperature optimum at

37 °C, pH optimum of 6.5, and the highest activity on gelatin and casein. On the

other hand, lipase from P. fluorescens 041 exhibits temperature optimum at 25 °C,

pH optimum of 7.5, and the highest activity on p-nitrophenyl palmitate.

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BURGER, M., WOODS, R.G., McCARTHY, C. BEACHAM, I.R. Temperature

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DRING, R., FOX, P.F. Purification and characterization of a heat-stable lipase from

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LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature, v. 227, p. 680-685, 1970.

LIAO, C.H., MCCALLUS, D.E. Biochemical and genetic characterization of an

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MAKHZOUM, A., OWUSU-APENTEN, R.K., KNAPP, J.S. Purification and

properties of lipase from Pseudomonas fluorescens strain 2D. International Dairy

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MARTINS, M.L., ARAÚJO, E.F., MANTOVANI, H.C., MORAES, C.A.,

VANETTI, M.C.D. Detection of the apr gene in proteolytic psychrotrophic bacteria

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CHAPTER 3

INVESTIGATION OF QUORUM SENSING IN STRAINS OF

Pseudomonas

fluorescens

ISOLATED FROM REFRIGERATED RAW MILK

3.1. INTRODUCTION

Gram-negative bacteria are the predominant psychrotrophic microflora

encountered in raw milk (URAZ and ÇITAK, 1998; DOGAN and BOOR, 2003),

with Pseudomonas spp. comprising at least, 50% of the total bacteria

(CHAMPAGNE et al., 1994). Many gram-negative bacteria, including members of

the genus Pseudomonas regulate gene expression in response to population density

by sensing the level of signal molecules produced and liberated into the environment

(SHAW et al., 1997). This phenomenon is termed quorum sensing and appears to be

a conserved process amongst prokaryotes since many bacteria have been shown to

use cell-cell communication to regulate diverse physiological processes (FUQUA et

al., 1996).

Several types of quorum sensing signals have been reported from different

P. fluorescens strains. Three different acyl-homoserine lactones (AHLs) were

detected in P. fluorescens 2-79 isolated from wheat by thin layer chromatography

(TLC) and mass spectrometry (MS) analysis, which were identified as N-(3-

hydroxyhexanoyl)-HSL, N-(3-hydroxyoctanoyl)-HSL, and N-(3-hydroxydecanoyl)-

HSL (SHAW et al., 1997). Later on, N-octanoyl-HSL and N-hexanoyl-HSL were

identified in this strain by Cha et al. (1998).

P. fluorescens F113 obtained from rhizosphere of sugarbeets produces N-(3-

hydroxy-7-cis-tetradecenoyl)-HSL, N-(decanoyl)-HSL, and N-(hexanoyl)-HSL

(LAUE et al., 2000). The gene hdtS capable of directing synthesis of all three P.

fluorescens F113 AHL does not belong to either of the known AHL synthase

families (LuxI or LuxM). However, Khan et al. (2005) showed that the phz operon of

P. fluorescens 2-79 is preceded by two genes, phzR and phzI, that are homologs of

quorum sensing gene pairs of the luxR-luxI family. Deleting phzR and phzI from

strain 2-79 led to loss of production of the antibiotics, as well as suite of AHLs.

El-Sayed et al. (2001) identified, when they studied the mupirocin antibiotic

biosynthetic cluster in a soil-borne bacterium, P. fluorescens NCIMB 10586, two

putative regulatory genes, mupR and mupI, whose predicted amino acid sequences

showed significant identity to proteins involved in quorum sensing dependent

regulatory systems such as LasR-LuxR and LasI-LuxI. This bacterium produced a

diffusible substance that overcomes the defect of a mupI mutant, and the use of

biosensor strains showed that MupI product can activate the P. aeruginosa lasR-lasI

system and that P. aeruginosa produces one or more compounds that can replace the

MupI product.

However, no AHL molecules were detected in P. fluorescens 1855.344, a

plant-growth-promoting rhizosphere bacterium (CHA et al., 1998), and in P.

fluorescens pf 7-14 isolated from rice rhizosphere (DUMENYO et al., 1998).

According to Cui et al. (2005), given the differences in AHL profiles from P.

fluorescens strains examined, it would clearly be of future interest to determine

whether there is any association between AHL profile and strain habitat. These

authors reported the identification of an AHL, N-(3-hydroxyoctanoyl)-HSL, in P.

fluorescens 5064 isolated from infected broccoli and the evidence for regulation of

biosurfactant production via this quorum sensing signal. AHL production appears to

be more common among plant-associated than among soilborne Pseudomonas spp.

(ELASRI et al., 2001).

Quorum sensing systems were involved in promoting cell attachment and

biofilm formation in P. fluorescens B52 isolated from milk, but these did not involve

short chain HSLs (ALLISON et al., 1998). Dunstall et al. (2005) evaluated nine AHL

compounds on lag phase duration and exponential growth rate of three strains of P.

fluorescens isolated from refrigerated raw milk. Two compounds N-

(benzoyloxycarbonyl)-L-homoserine lactone and N-(3-oxyhexanoyl)-DL-homoserine

lactone were found to significantly (p<0.001) reduce the lag phase duration and

increase the exponential growth rate of these strains. However, P. fluoresecens

strains isolated from cooled raw milk induced only the monitor strains of

Agrobacterium tumefaciens ultrasensitive for AHL (PINTO et al., 2007).

A novel family of signaling compounds identified as diketopiperazines

(DKPs) has been discovered in cell-free supernatants of P. aeruginosa, P.

fluorescens, Pseudomonas alcaligenes, Enterobacter agglomerans and Citrobacter

freundii (HOLDEN et al., 1999). Although there is abundant literature data about

dipeptides isolated from microbial sources their true origin remains controversial

(HERNÁNDEZ et al., 2004). However, several dipeptides isolated from

microorganisms display relevant biological activities (HOLDEN et al., 1999;

HOLDEN et al., 2000; HERNÁNDEZ et al., 2004). Microbial DKPs found to date

appear to be catabolic products of peptone or other components of the nutrient rich

media (PRASAD, 1995).

While the AHLs are confined to a reasonably narrow range of bacteria, recent

evidence has suggested the existence of a universal quorum sensing language

(CÁMARA et al., 2002) known as a family of molecules termed AI-2, common to

many gram-negative and gram-positive bacteria, including some pathogens

(MILLER and BASSLER, 2001). However, there is no direct evidence for the

involvement of AI-2 in regulation of pathogenic characteristics. AI-2 has evolved

several diverse species specific roles while simultaneously remaining a universal

signal recognizable across numerous species of bacteria (DeLISA et al., 2002). This

autoinducer and its biosynthetic via are the same among all species that have luxS

(MOK et al., 2003).

Considering that some strains of P. fluorescens use the quorum sensing

mechanism to regulate antibiotic production and other phenotypes, and considering

that expression of spoilage enzymes occurs in high population density, this research

focused on elucidation of quorum sensing in strains of P. fluorescens isolated from

cooled raw milk.

3.2. MATERIAL AND METHODS

3.2.1. Bacterial strains and growth conditions

The strains used in the present study are listed in Table 1. Unless otherwise

stated, P. fluorescens was grown at 25 °C and other strains were grown at 30 °C in

Luria-Bertani (LB) medium (ANDERSEN et al., 1998) or AB minimal medium

(CLARK and MAALOE, 1967) supplemented with 10 mM citrate (ABC). Vibrio

harveyi strains were grown at 30 °C in AB Vibrio medium (BASSLER et al., 1994).

Solid media were routinely solidified with 1.4% agar, while growth media for

examination of swarming motility contained 0.4% (w/v) agar (EBERL et al., 1996).

Antibiotics were added as required at final concentrations of 20 µg/ml for gentamicin

and tetracycline, 50 µg/ml for trimethroprim and spectinomycin, 100 µg/ml for

ampicillin, and 10 µg/ml for chloramphenicol. Kanamycin was used at 30 µg/ml for

V. harveyi and at 50 µg/ml for E. coli S17-1 and P. fluorescens. Besides, tellurite, an

inhibitor agent, was used at 100 µg/ml. Growth of liquid cultures was monitored

spectrophotometrically with an Ultrospec 3100 Pro spectrophotometer (Biochrom,

Ltd., Cambridge, England) by measurement of the optical density at 600 nm.

Table 1 – Bacterial strains and plasmids used in this study

Strain Plasmid Description Reference or source

Agrobacterium

tumefaciens A136

pCF373, pCF218,

, Spc

Monitor strain: detects AHL with 3-

oxo, 3-hydroxy, and 3-unsubstituted

side chain

Fuqua and Winans,

1996; Shaw et al.,

1997

A. tumefaciens KYC55

pJZ410, pJZ372,

pJZ384, Tc

, Spc

Ultrasensitive strain: detects many

diverse AHLs at extremely low

concentrations

Zu et al., 2003

A. tumefaciens NTL4 pZLR4, Gm

Monitor strain: detects AHL with 3-

oxo, 3-hydroxy, and 3-unsubstituted

side chain

Cha et al., 1998

Burkholderia cepacia

H111

Positive control in the cross-streak to

E. coli pSB403, and P. putida F117

pAS-C8

Riedel et al., 2003

Burkholderia

vietinamensis

Positive control in the cross-streak to

P. putida F117 pKR-C12

Wopperer et al., 2006

Chromobacterium

violaceum CV026

Monitor strain: detects AHL

compounds with unsubstituted side

chains from C4 to C8 in length.

Ravn et al., 2001

Escherichia

coli HB101

pRK600, Cm

Helper

Laboratory of

Microbiology,

University of Zürich

E. coli MT102 pSB403, Tc

Monitor strain: exhibits the highest

sensitivity for 3-oxo-C6-HSL.

However, several other AHL

molecules are detected by this sensor.

Winson et al., 1998;

Gotschlich et al.,

2001; Steidle et al.,

2001

E. coli XL1-Blue

pMLBAD-aiiA-

Donor of pMLBAD-aiiA, Gm

that

codify the lactonase enzyme

This study

E. coli XL1-Blue

pMLBAD-aiiA-

Tel

Donor of pMLBAD-aiiA, Tel

that

codify the lactonase enzyme

Laboratory of

Microbiology,

University of Zürich

E. coli XL1-Blue

pMLBAD-aiiA-

Trm

Donor of pMLBAD-aiiA, Trm

that

codify the lactonase enzyme

Wopperer et al., 2006

Pseudomonas

aeruginosa PAO1

Positive control in the cross-streak to

C. violaceum CV026, A. tumefaciens

NTL4, and A. tumefaciens A136

Laboratory of

Microbiology,

University of Zürich

Pseudomonas

fluorescens 07A

Wild type Martins et al., 2005

P. fluorescens 07A-2

pMLBAD-aiiA-

Trm

-Gm

Transconjugant, express the lactonase

enzyme

This study

P. fluorescens 041 Wild type Martins et al., 2005

P. fluorescens 041-3

pMLBAD-aiiA-

Trm

-Gm

Transconjugant, express the lactonase

enzyme

This study

P. fluorescens 097 Wild type Martins et al., 2005

P. fluorescens 0109 Wild type

Pinto, 2004; Pinto et

al., 2007

Pseudomonas putida

F117

pAS-C8, Gm

Monitor strain: exhibits the highest

sensitivity for OHL

Wopperer et al., 2006

P. putida F117 pKR-C12, Gm

Monitor strain: it detects 3-oxo-C12-

and 3-oxo-C10-HSL

Wopperer et al., 2006

Vibrio harveyi BB170 Monitor strain: detects AI2 Schauder et al., 2001

V. harveyi BB120 Positive control: AI2 producer Schauder et al., 2001

3.2.2. Detection and quantification of signal molecules

AHL production was investigated by cross-streaking P. fluorescens strains

against E. coli MT102 pSB403, C. violaceum CV026, A. tumefaciens NTL4, and A.

tumefaciens A136. The cross-streak of P. fluorescens against E. coli MT102 and C.

violaceum was done on LB agar plates, whereas the cross-streak of P. fluorescens

against A. tumefaciens strains was done on LB agar plates supplemented with 80

μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Also the A.

tumefaciens strains were cross-streaked against themselves in AB medium. After

incubation up to 2 days at 30 °C, bioluminescence produced by E. coli MT102 was

detected into a dark box containing a highly sensitive photon-counting camera

(C2400-40; Hamamatsu Photonics Herrsching, Germany) (STEIDLE et al., 2001).

Violacein production by C. violaceum CV026 was detected as violet pigments in the

medium and β-galactosidase activity was identified by forming blue pigments. These

results were documented by photographing the plates.

Pseudomonas putida F117 harbour a plasmid pKR-C12, which contains a

PlasB-gfp(ASV) translational fusion, together with the lasR gene placed under

control of Plac. This sensor strain is highly sensitive for 3-oxo-C12- and 3-oxo-C10-

HSL. Plasmid pAS-C8 was constructed from components of the cep system of

Burkholderia cenocepacia H111 and contains a PcepI-gfp(ASV) translational fusion

together with the cepR gene transcribed from the Plac promoter of the broad-host-

range plasmid pBBR1MCS-5. This sensor plasmid responds very efficiently to C8-

HSL and only with low efficiency to other AHL molecules. After overnight

incubation of cross-streaking plates at 30 °C, fluorescence was detected by

illumination with blue light by using an HQ 480/40 filter (AHF-Analysentechnik,

Tübingen, Germany) in combination with a halogen lamp (Volpi, Schlieren,

Switzerland) as a light source in a dark box equipped with a light-sensitive camera

(Hamamatsu Photonics, Herrsching, Germany) with a Pentax CCTV camera lens and

an HQ 535/20 filter.

Since E. coli MT102 pSB403 is able to detect low amount of AHL, it was

used for quantification of these molecules. The plasmid pSB403 contains the

Photobacterium fischeri luxR gene together with luxI promoter region as a

transcriptional fusion to bioluminescence genes luxCDABE of Photorhabdus

luminescens. The quorum sensing system of P. fischeri relies on 3-oxo-C6-HSL, and

the sensor plasmid consequently exhibits the highest sensitivity for this AHL

molecule. However, several other AHL molecules are detected by the sensor, albeit

with some what reduced sensitivity (WINSON et al. 1998). A volume of 1 ml of

overnight culture of E. coli MT102 pSB403 was inoculated into 5 ml LB

supplemented with tetracycline and incubated at 30 °C for 1 h. Then, 100 µl of filter-

sterilized culture supernatants in LB of P. fluorescens were added to 100 µl of an

exponential culture of the sensor strain in the wells of a microtiter dish. After

incubation at 30 °C for 3 h, the expression of the bioluminescence reporter genes was

measured using the program KC4 (Bio-Tek Instruments, Highland Park, Box 998,

Vermont, USA). AHL concentrations were determined by comparing

bioluminescence signal intensities with a defined concentration (0.1 mg/ml) of pure

3-oxo-C6-HSL.

3.2.3. Extraction of quorum sensing signal from supernatants

P. fluorescens 07A and 041 (10

CFU/ml) was inoculated in 600 ml of LB,

TYEP, King’s B, and AB minimal medium supplemented with 10 mM citrate

(ABC). The cultures were incubated with aeration at 25 °C for 20 h or until the

population reach 10

CFU/ml. Then, the cells were harvested by centrifugation at

10,000 g for 20 min at 4 °C and 250 ml of the cell free supernatants were mixed with

100 ml of dichloromethane stabilized with ethanol in a 1,000 ml separating funnel.

The mixture was shacked for 3 min with aeraton every 20 s. When the two phases

were separated, the dichloromethane-phase was collected (lower phase). The upper

phase (aqueous phase) was mixed with 100 ml of dichloromethane and shaked again

as described above. Lower dichloromethane-phase was collected and mixed with the

first one. These steps were repeated until finishing the 600 ml of supernatant. Then,

the remaining water was removed with water free MgSO

and it was filtrated using

Whatman paper. The filtered extracts were concentrated in a rotary evaporator at 40

°C, resuspended in 250 µl ethyl acetate, and maintained at -20 °C.

3.2.4. Detection of signal molecules in supernatant of P. fluorescens

Thirty milliliter of overnight culture of E. coli MT102 pSB403 were

inoculated in 150 ml of LB agar. The inoculated LB plates were solidified and 6 µL

of AHL extracts obtained from the supernatant of King’s B, LB, and TYEP

inoculated with P. fluorescens 07A were transferred as drops to the plate’s surface.

Aliquots of 0.6 µL of HHL 1 mg/ml were used as positive controls. The plates were

incubated overnight and the activation of the AHL monitor strain E. coli MT102

pSB403 was observed into a dark box that contained a highly sensitive photon-

counting camera (C2400-40; Hamamatsu Photonics Herrsching, Germany) as

described by Steidle et al. (2001).

3.2.5. Detection of signal molecules using Thin Layer Chromatography (TLC)

A line of 1.5 cm afar from the borders of the TLC plate (C

reversed-phase

thin-layer plate, hydrocarbon impregnated silica gel, RPS 20 x 20 cm, Analtech-

uniplate

) was signed and aliquots from 10 to 20 µl of the ethyl acetate extracts

were loaded on TLC plate. The extract was loaded on TLC drop by drop of 2 µl and

dried in cold air step by step. TLC plates were developed with 60% (v/v) methanol-

water solvent mixture in a saturated glass chamber. After elution, the plates were

dried in cold air.

LB soft agar (150 ml) at 42 °C was mixed with 30 ml of the activated monitor

strains E. coli MT102 (pSB403), C. violaceum CV026, or A. tumefaciens NTL4

according to the AHLs to be detect. For A. tumefaciens NTL4, 30 ml of this culture

was mixed with 110 ml AB soft agar added of 80 µg/ml X-Gal (5-bromo-4-chloro-3-

indolyl-β-D-galactopyranoside). After supplementation with the appropriated

monitor strain, the soft agar was dispensed on a dried TLC plate to produce a 2 to 3

mm thick layer. After 20 min, the plate was put in an airproof box with a wet paper

inside and incubated overnight at 30 °C.

The documentation system was dependent on the monitor strain used. When

C. violaceum CV026 was used as a monitor, the signal molecules could be identified

by forming violet pigments after 48 h incubation. For E. coli MT102 pSB403, after

incubation overnight at 30 °C, the TLC plates were put into a dark box and

bioluminescence was detected with a highly sensitive photon-counting camera

(C2400-40; Hamamatsu Photonics Herrsching, Germany). For A. tumefaciens NTL4,

the material was incubated until 48 h and AHLs could be identified by visualizing

blue pigments. In all cases the documentation was done by photographing.

3.2.6. DNA manipulations, PCR reactions and sequencing

3.2.6.1. DNA manipulations

All DNA manipulations were developed as described in chapter 2, item 2.2.6.1.

3.2.6.2. Amplification of the AHL synthase (

phzI

and

mupI

) genes of

fluorescens

by PCR

The PCR reaction consisted of 2.0 mM MgCl

, 5.0 µl of 10X buffer Ex Taq,

2.5 mM deoxynucleotide triphosphates (dNTPs), 25 pmol of each primer, 1 U Ex

Taq DNA polymerase, and 40 ng of DNA of P. fluorescens 07A and 041 in a final

volume of 50 µl. Primers based on the sequences of the phzI (GenBank accession

number

L48616) and mupI gene (GenBank accession number AF318063) of P.

fluorescens were constructed (Table 2) and synthesized by Microsynth (Zürich,

Switzerland). PCR reactions were carried out in a T3 thermocycler (Biometra

Biolabo Scientific Instruments, Zürich, Switzerland).

Table 2 – Primers used to amplify the phzI and mupI genes by PCR

Primer Sequence (5’-3’) Application

phzI-F ATG CAC ATG GAA GAG CAC phzI gene

phzI-R GAG TTT GAT GGC GAG GAT phzI gene

phzI-F new GAA TGG GAT CAA TAC GAC AC phzI gene

phzI-F new-1 TTC ACC ACC CGC GAA CCG C phzI gene

phzI-R new GCC GAG AGT TTG ATG GCG AGG phzI gene

mup-F TAA TAG ACA AAC GCG AGA A mupI gene

mup-R GTT AAC TTC AAC AGC GAT G mupI gene

3.2.6.3. Sequencing of the AHL synthase genes

The M13 Forward and Reverse primers were used to sequence the fragments

of phzI and mupI genes cloned into pCR2.1-TOPO.

3.2.7. Evaluation of P. fluorescens resistance against different antibiotics and

tellurite

P. fluorescens strains were inoculated into 5 ml of LB broth containing 100

µg/ml tellurite or different antibiotics (100 µg/ml trimethroprim, 20 µg/ml

chloramphenicol, 25 µg/ml gentamicin, 100 µg/ml ampicillin, 50 µg/ml kanamycin,

20 µg/ml tetracycline, and 50 µg/ml spectinomycin). Growth at 25 °C was observed

at 600 nm after incubation for 48 h.

3.2.8. Cloning of the gentamicin-3-acetyltransferase gene on broad-host-range

expression vector

The gentamicin-3-acetyltransferase gene (GenBank accession number

U25061) of pBBR1MCS-5 was amplified using the primer pair Gem-F (5’ ATT

ATG CAT GAA CCT GAA TCG CCA GCG G 3’) and Gem-R (5’ ATT ATG CAT

GTT GAA CGA ATT GTT AGG TGG C 3’). The introduced restriction site NsiI is

underlined. The amplicon was digested with NsiI and ligated directionally into the

broadhost-range expression vector pMLBAD-aiiA-Trm

(WOPPERER et al., 2006)

cut with the same enzyme, yielding pMLBAD-aiiA-Trm

-Gm

. This plasmid

containing the aiiA gene which encodes the lactonase enzyme was transferred to E.

coli XL1-Blue by transformation.

3.2.9. Conjugative plasmid transfer

Plasmids were delivered to P. fluorescens strains by triparental mating as

described previously (DE LORENZO and TIMMIS, 1994). Briefly, donor (E. coli

XL1-Blue pMLBAD-aiiA-Trm

-Gm

) and recipient strains, as well as the helper

strain E. coli HB101 (pRK600), were grown overnight in 5 ml of LB medium

supplied with the appropriate antibiotics. After subculturing to an optical density of

0.9 at 600 nm, the cells from 2 ml of the culture were harvested, washed, and

resuspended in 500 µl of LB medium. Donor and helper cells (100 µl each) were

mixed and incubated for 10 min at room temperature. Then, 200 µl of the recipient

cells was added and the mixture was spot inoculated onto the surfaces of pre-heated

LB agar plates. After overnight incubation at 30 °C, the cells were plated on

Pseudomonas Isolation Agar (PIA) (Becton Dickinson Biosciences, Sparks, MD)

containing antibiotics for counter selection of the donor, helper, and untransformed

recipient cells.

After identity confirmation of the transconjugants, quorum sensing signals

were extracted from supernatants of different media as described in chapter 3, item

3.2.3, and the detection of signal molecules by Thin Layer Chromatography (TLC)

were developed as described in chapter 3, item 3.2.5.

3.2.10. Phenotypic characterization of wild type and transconjugant strains

Biofilm formation in polystyrene microtiter dishes was assayed essentially as

described previously (PRATT and KOLTER, 1998) with a few modifications. Cells

of P. fluorescens 07A and 041 wild-type and transcojugant were grown in the wells

of microtiter dishes in 100 µl of LB, minimal medium salt (MMS) or AB medium

supplemented with 10 mM citrate (ABC) for 48 h at 25 °C. Thereafter, the medium

was removed, and 100 µl of a 1% (wt/vol) aqueous solution of crystal violet (CV)

was added. After staining at room temperature for 20 min, the dye was removed, and

the wells were washed thoroughly. For quantification of attached cells, the CV was

solubilized in an 800:120 (vol/vol) mixture of ethanol and dimethyl sulfoxide, and

the absorbance was determined at 570 nm.

The ability to form a swarming colony was tested by point inoculating strains

into ABC minimal medium supplemented with 0.1% Casamino Acids and solidified

with 0.4% agar as previously described (HUBER et al., 2001).

Proteolytic activity was determined by streaking strains on LB agar

supplemented with 2% (w/v) skim milk (WOPPERER et al., 2006), and in cultures

of P. fluorescens in ABC, MMS, and TYEP using azocasein assay as described in

chapter 2, item 2.2.3.2.

3.2.11. Identification of signal molecules by mass spectrometry

Culture supernatant of TYEP medium was extracted as described in chapter 3

item 3.2.3 using ultra pure dichloromethane stabilized with ethanol and analyzed by

gas chromatograph-mass spectrometry (GC-MS).

3.2.12. Detection of AI-2 in supernatant of LB medium inoculated with P.

fluorescens

P. fluorescens 07A, 041, 097, and 0109 were grown overnight with aeration

at 28 °C on LB medium. Cell-free culture supernatants were prepared by removing

the cells from the growth medium by centrifugation at 10,000 g for 20 min. The

cleared culture supernatants were passed through 0.2 μm filters and stored at -20 °C.

Vibrio harveyi BB120 was used as a positive control, and it was grown overnight at

30 °C with aeration in AB Vibrio medium. Cell-free culture fluids from V. harveyi

BB120 were prepared from overnight culture exactly as described before for P.

fluorescens. Aliquots of 10 μl of cell-free culture fluids were added to 96-well

microtiter dishes. The monitor strain, V. harveyi BB170, was grown with aeration for

16 hours at 30 °C in AB medium and diluted 1:5,000 into fresh AB medium.

Aliquots of 90 μl of diluted cells were added to wells containing the P. fluorescens

cell-free culture fluids. Positive control wells contained 10 μl of cell-free culture

fluid from V. harveyi BB120 and negative control wells contained 10 μl of sterile

growth medium (LB or AB). Microtiter dishes were shaken in a rotary shaker at 175

RPM at 30 °C. Bioluminescence was measured using the program KC4 (Bio-Tek

Instruments, Highland Park, Box 998, Vermont, USA).

3.3. RESULTS AND DISCUSSION

3.3.1. Detection of signal molecules produced by P. fluorescens

P. fluorescens strains isolated from refrigerated raw milk did not induce the

biosensor strains C. violaceum CV026, E. coli MT102 pSB403, P. putida F117 pAS-

C8, and P. putida F117 pKR-C12 (Table 3).

Table 3 – Activation of the AHL monitor strains in cross-streak experiments

Result obtained with Bacteria

CV 026 pSB403 F117 (pAS-C8) F117 (pKR-C12) A 136 NTL4

P. fluorescens 07A - - - - + +

P. fluorescens 041 - - - - + +

P. fluorescens 097 - - - - + +

P. fluorescens 0109 - - - - + +

B. cepacia H111 Nd +++ +++ Nd Nd Nd

B. vietinamensis Nd Nd Nd +++ Nd Nd

P. aeruginosa PAO1 +++ Nd Nd Nd +++ +++

The six monitor strains were cross-streaked against P. fluorescens on LB agar plates. Following up to

48 h of incubation at 30

C, the production of violacein by C. violaceum CV026, bioluminescence by

E. coli pSB403, green fluorescent protein gfp(ASV) by P. putida F117, and β-galactosidase activity

by A. tumefaciens A136 and NTL4 was visualized as described in material and methods. Levels of

activation are indicated as follows: +++: strong activation, diffusion of AHL > 1 cm; +: activation,

diffusion of signal of 0.3 cm; -: no detectable activation. Nd: not determined.

However, they were able to induce weakly A. tumefaciens A136 and NTL4

(Table 3) which indicates that signal molecule(s) produced by P. fluorescens strains

were in low concentration or they are different from the cognate autoinducer that

activates TraR protein in A. tumefaciens. Once A. tumefaciens strains responded to

substances present into culture media, a false positive result should be considered,

since in some experiments these strains were able to induce themselves (Figure 1).

Other molecules besides AHLs such as diketopiperazines (DKPs) could activate

biosensors, underlining the importance of chemical characterization of the molecules

identified in such bioassays (HOLDEN et al., 1999). The development and use of

ultrasensitive biosensors must be done with caution once the reality of the results

obtained can be compromised because they can detect compounds that are not used

as signal molecule in nature. The biological significance of these compounds like

cyclic dipeptides (diketopiperazines) as putative signal molecules is discussed, with

evidence presented that these compounds are capable of activating or antagonizing

lux-based AHL biosensors and AHL-dependent phenotypes (HOLDEN et al., 1999).

Previously, many Pseudomonas spp. have been shown to produce and secrete

signal molecules such as AHLs (SHAW et al., 1997; CHA et al., 1998; KIEVIT and

IGLEWSKI, 2000; LAUE et al., 2000; PARSEK and GREENBERG, 2000;

ANDERSEN et al., 2001; EL-SAYED et al., 2001; STEIDLE et al., 2001;

WHITEHEAD et al., 2001; CUI et al., 2005; JUHAS et al., 2005; KHAN et al.,

2005). Cui et al. (2005) suggested that AHL production seems to be more common

among P. fluorescens closely associated with plants than among their soilborn

counterparts. Because of the specificity requirements of the R protein, most of the

detection systems are limited in the range of AHLs to which they respond (SHAW et

al., 1997). Therefore, it represents a limitation of the bioassay since bacteria often

produce more than one AHL molecule. This limitation can be overcome by using

multiple monitor systems (PINTO et al., 2007).

Figure 1 – Activation of the AHL monitor strain A. tumefaciens in cross-streak

experiments in AB medium after 48 h at 30 °C. A) A. tumefaciens

NTL4 in cross-streak to A. tumefaciens NTL4; B) A. tumefaciens

KYC55 in cross-streak to A. tumefaciens KYC55; C) A. tumefaciens

A136 in cross-streak to A. tumefaciens A136.

3.3.2. Detection of bioluminescence induced by P. fluorescens

Signal molecule extract obtained from P. fluorescens 07A and 041 did not

induce E. coli MT102 pSB403 in the assay developed to detect the bioluminescence

production (Table 4). Therefore, this result suggests that P. fluorescens 07A and 041

isolated from cooled raw milk did not produce AHLs able to induce a high sensitive

biosensor as E. coli MT102 pSB403 and reinforce that the molecules able to induce

A. tumefaciens in the cross-streak assays are other compounds present in the growth

media. According to Winson et al. (1998), there is a significant advantage of using

lux sensors since the sensitivity to AHL is in a range of picomol and nanomol

concentrations over a large linear range in real time. By combining the results from

lux sensor the activity profiles can be compared with those of known standards to

give a preliminary identification of the nature of the AHL under investigation. This

information can then be used to aid the development of appropriate extraction and

identification procedures.

Table 4 – Values of bioluminescence produced by E. coli MT102 pSB403 at 175 nm

after growth in LB broth supplemented with supernatant of P. fluorescens

and supplemented with 3-oxo-C6-HSL. Data represent average of

triplicate experiments.

Dilution rate P. fluorescens

07A

P. fluorescens

041

Negative control

3-oxo-C6-HSL

Positive control

1/2 13532 15823 14820 Nd*

1/4 15261 13831 15003 Nd

1/8 13967 15360 13230 Nd

1/16 14401 16580 15340 Nd

1/32 16977 16036 16720 51802

1/64 15862 14159 14579 28723

1/128 17092 17619 16220 21817

1/256 15716 13420 14943 20942

*Nd – not detected. The intensity of the signal was higher than the limit of detection of the equipment.

3.3.3. Supplementation of LB inoculated with E. coli MT102 pSB403 with

extracts obtained from different media

As no activity of signal molecules was found in the supernatant of LB

medium inoculated with P. fluorescens 07A and 041 against E. coli MT102 pSB403

using the microtiter dishes assay, it was tried to find correlation between the growth

media and the production of signal compounds, but no influence on AHL production

was found (Figure 2).

However, Mcphee (2001) found that synthesis of AHLs by Pseudomonas was

influenced by the composition of the growth medium. It was suggested that AHL

production is not only influenced by cell density, but environmental factors such as

growth medium also are important. According to Mcphee (2001), P. fluorescens was

found to up-regulate enzyme synthesis when it grew in a spent culture supernatant,

presumably having already high levels of synthesized AHL.

Figure 2 – Activation of the AHL monitor strain E. coli MT102 pSB403 after

supplementation of the medium with 6 µL of signal molecule extracts

obtained from the supernatant of different media inoculated with P.

fluorescens 07A. The plate was incubated at 30 °C for 18 h.

3.3.4. Detection of signal molecules using TLC

The extracts of different media inoculated with P. fluorescens 07A and 041

induced A. tumefaciens NTL4 when it was used as a reporter strain on TLCs (Figures

3, 4, 5, 6 and 7). The only exception was observed in ABC minimal medium

inoculated with P. fluorescens 07A wild type and transconjugant (Figure 3). This

result confirms that in a pour medium, which contains only minerals and the carbon

source was sterilized by filtration, compounds like DKPs is not produced. Then,

there are no molecules able to induce A. tumefaciens.

Besides, AHL biosensors as E. coli MT102 pSB403 and C. violaceum CV026

did not detect any signal from the extracts obtained from LB medium inoculated with

P. fluorescens 07A and 041.

Extracts obtained from different media without inoculation that were used as

a negative control were able to induce A. tumefaciens NTL4 (Figures 4B, 5AB, 6,

and 7AB). However, it can be verified that the extract of culture of P. fluorescens

resulted in a signal with intensity and position on TLC plates different from the

signal obtained with extracts of negative controls (Figures 4B, 5AB, 6, and 7AB).

This indicates that P. fluorescens might produce some substance that is able to

induce A. tumefaciens, but this substance needs to be characterized to confirm its

identity and its biological potential as an autoinducer compound.

C6-HSL

King’s B

TYEP

Figure 3 - A representative thin-layer chromatogram of the signal molecules present

in cell free supernatants of P. fluorescens 07A. The spots were detected

with the A. tumefaciens NTL4 reporter strain. Standards: N-(3-

oxohexanoyl)-L-homoserine lactone (OHHL); N-(octanoyl)-L-

homoserine lactone (OHL); and N-(hexanoyl)-L-homoserine lactone

(HHL).

Figure 4 - Representative thin-layer chromatograms of the signal molecules present

in cell free supernatants of P. fluorescens 07A. The spots were detected

with A. tumefaciens NTL4 reporter strain. Standards: N-(3-oxohexanoyl)-

L-homoserine lactone (OHHL); N-(octanoyl)-L-homoserine lactone

(OHL); and N-(hexanoyl)-L-homoserine lactone (HHL); (NC) negative

control, extract from LB not inoculated. (A) 10 µl of extract obtained

from LB medium. (B) 20 µl of extract obtained from LB medium.

OHHL 07A 07A-aiiA 07A OHL HHL

ABC medium TYEP medium

10 µl 10 µl 10 µl

OHHL 07A 07A-aiiA NC HHL OHL

LB medium

20 µl 20 µl 20 µl

OHHL 07A 07A-aiiA NC HHL OHL

LB medium

10 µl 10 µl 10 µl

Figure 5 - Representative thin-layer chromatograms of the signal molecules present

in cell free supernatants of P. fluorescens 041. The spots were detected

with the A. tumefaciens NTL4 reporter strain. Standards: N-(3-

oxohexanoyl)-L-homoserine lactone (OHHL); N-(hexanoyl)-DL-

homoserine lactone (HHL); N-(octanoyl)-L-homoserine lactone (OHL);

(NC) negative control, extract from LB not inoculated. (A) 10 µl of

extract obtained from LB medium. (B) 20 µl of extract obtained from LB

medium.

Figure 6 - A representative thin-layer chromatogram of the signal molecules present

in cell free supernatants of P. fluorescens 07A and 041. The spots were

detected with the A. tumefaciens NTL4 reporter strain. Standards: N-(3-

oxohexanoyl)-L-homoserine lactone (OHHL); N-(hexanoyl)-L-

homoserine lactone (HHL); N-(octanoyl)-L-homoserine lactone (OHL);

(NC) negative control, extract from LB not inoculated.

OHHL 041 041-aiiA NC

LB medium

20 µl 20 µl 20 µl

OHHL 041-aiiA 041 NC HHL OHL

LB medium

10 µl 10 µl 10 µl

OHHL 07A 041 NC HHL OHL

King’s B medium

10 µl 10 µl 10 µl

Figure 7 - Representative thin-layer chromatograms of signal molecules present in

cell free supernatants of P. fluorescens 07A and 041. The spots were

detected with A. tumefaciens NTL4 reporter strain. Standards: N-(3-

oxohexanoyl)-L-homoserine lactone (OHHL); N-(hexanoyl)-L-

homoserine lactone (HHL); N-(octanoyl)-L-homoserine lactone (OHL);

(NC) negative control, extract from LB not inoculated. (A) 10 µl of

extract obtained from TYEP medium. (B) 10 µl or 20 µl of extract

obtained from TYEP medium. The sample used in the last TLC was

obtained using ultra pure dichloromethane and characterized by gas

chromatograph-mass spectrometry.

The involvement of quorum sensing in growth of P. fluorescens was

suggested by Whan et al. (2000). N-benzoyloxycarbonyl-L-homoserine lactone and

N-3-oxyhexanoyl-DL-homoserine lactone significantly reduced the lag phase

duration and increased the exponential growth rate of three strains of P. fluorescens

(DUNSTALL et al., 2005). However, no signal molecules were found on TLC by

Pinto et al. (2007) and by Viana (2006).

3.3.5. Amplification and sequencing of phzI and mupI genes by PCR

Since El-Sayed et al. (2001) and Khan et al. (2005) pointed out a relationship

between the quorum sensing mechanism and antibiotic production by P. fluorescens

NCIMB 10586 and P. fluorescens 2-79, and characterized the AHL synthases as

MupI and PhzI, respectively, primers based on these sequences were developed and

OHHL 07A NC 07A NC OHL/HHL

TYEP medium

10 µl 10 µl 20 µl 20 µl

OHHL 07A 041 NC HHL OHL

TYEP medium

10 µl 10 µl 10 µl

used to amplify these genes by PCR in P. fluorescens 07A and 041. It was obtained

unspecific PCR products when it was used primers to amplify mupI and phzI genes.

However, bands of right size (530 bp for mupI and 510 bp for phzI) were extracted

from the gels, cloned into pCR2.1-TOPO and sequenced. The results obtained were

sequences of P. fluorescens not related to any AHL synthase, confirming that these

strains 07A and 041 isolated from cooled raw milk do not have genes homologues to

phzI and mupI.

3.3.6. Resistance of P. fluorescens against some antibiotics and selective agent

Although P. fluorescens strains isolated from raw milk were not able to grow

in LB supplemented with tellurite, a selective agent, and in LB supplemented with

gentamicin (Table 5), they grew well in LB supplemented with other antibiotics. The

wide spectra of antibiotic resistance observed among P. fluorescens (Table 5) may

demonstrate high selective pressure into raw milk. This datum confirms the data

obtained by other authors who observed a high patter of antibiotic resistance among

bacteria isolated from milk in Brazil (ARAÚJO, 1998; CARNEIRO and JÚNIOR,

2006).

Table 5 – Tellurite and antibiotic susceptibility of Pseudomonas fluorescens in LB

broth

P. fluorescens Antibiotic

07A 041 097 0109

Tellurite - - - -

Trimethoprim + + + +

Chloramphenicol + + + +

Gentamicin - - - -

Ampicillin + + + +

Kanamycin + + + +

Tetracycline + + + +

Spectinomycin + + + +

Growth is indicated as follows: +, growth; -, no detectable growth.

3.3.7. Cloning of gentamicin-3-acetyltransferase gene in pMLBAD-aiiA-Trm

and mobilization to P. fluorescens 07A and 041

Since P. fluorescens 07A and 041 were able to grow in LB supplemented

with many antibiotics, it was necessary to obtain a new donor strain. First, E. coli

XL1-Blue pMLBAD-aiiA-Tel

was used as donor, but as P. fluorescens

transconjugant was not able to grow well in LB supplemented with tellurite, it was

necessary to clone the gentamicin-3-acetyltransferase gene in pMLBAD-aiiA-Trm

To express aiiA lactonase gene in P. fluorescens strains, a fragment of

gentamicin-3-acetyltransferase gene (860 bp) was amplified by PCR as described in

Material and Methods and cloned directionally into the NsiI site (Figure 8) of a

broad-host-range expression vector pMLBAD-Trm

(LEFEBRE and VALVANO,

2002) that had the aiiA gene (797 bp) cloned into NcoI and HindIII sites by

Wopperer et al. (2006), yielding pMLBAD-aiiA-Trm

-Gem

. In pMLBAD-aiiA-

Trm

-Gem

, the aiiA gene is transcribed from P

BAD

promoter of E. coli, and thus

expression is inducible with arabinose. Thus, routinely it was used 0.02% arabinose

in all further experiments, as described by Wopperer et al. (2006).

To test the functionality of the construct and the applicability of the quorum-

quenching approach, pMLBAD-aiiA-Trm

-Gem

was conjugated into P. fluorescens.

To confirm that P. fluorescens strains contained the plasmid with the right inserts,

this plasmid was extracted, and restricted with NcoI and HindIII (Figure 9).

It was observed that the signal extract obtained from strains of P. fluorescens

transconjugant inoculated in LB induced A. tumefaciens NTL4 (Figure 4AB, and

5AB). This data confirm that the molecule(s) that induced A. tumefaciens was not

compromised by lactonase expression.

Figure 8 – Map of pMLBAD showing the NcoI and HindIII sites used by Wopperer

et al. (2006) to clone the aiiA gene and the site to NsiI used to clone

gentamicin-3-acetyltransferase gene (this work). dhfr, dihydrofolate

redutase gene encoding trimethoprim resistance; P

BAD

, arabinose-

inducible promoter; araC, transcriptional regulator gene; rrnB,

transcriptional terminator; MCS, multiple cloning site; mob, gene

required for conjugal transfer of plasmid; rep, replication protein gene;

ori, origin of replication. Adapted from Lefebre and Valvano (2002).

Figure 9 – Agarose gel electrophoresis of restriction reactions of pMLBAD-aiiA-

Trm

-Gem

containing the aiiA and gentamicin-3-acetyltransferase

genes cloned. Line 1, Standard; line 2, P. fluorescens 07A-2

transconjugant; line 3, P. fluorescens 041-3 transconjugant.

NsiI

500

1000

1 2 3

10000

2000

250

750

1500

pMLBAD-Trm

aiiA

Gentamicin gene restricted with NcoI

3.3.8. Phenotypic characteristics of P. fluorescens wild type and transconjugants

3.3.8.1. Biofilm

After 48 hours of incubation, it was observed that P. fluorescens 07A and 041

produced less biofilm in LB and MMS than in ABC medium. The strain 041 was

able to bind better than 07A in polystyrene microtiter dishes (Figure 10). Viana

(2006) also observed that different strains of P. fluorescens had different ability to

bind in polystyrene and that minimal medium enhanced attachment. The ABC

minimal medium is rich in divalent ions as phosphate, Ca

, Mg

, and Fe

According to Fletcher et al. (1988), divalent ions as Ca

and Mg

can influence

directly biofilm formation due to electrostatic interactions, and indirectly via process

of adhesion dependent of microorganism physiology because they can act as

cofactors of enzymes. The presence of ions as Ca

improve cross-binding between

cells and between cells and surfaces (KOERSTGENS et al., 2001). Maybe, because

the abundance of these compounds in ABC minimal medium, better biofilm

formation in this medium was observed compared to LB and MMS.

It was not verified significant difference (p>0.01) on the ability to produce

biofilm when wild type strains were compared to transconjugants (Figure 10). This

result shows that the quorum quenching mechanism did not influence this phenotype

in these strains of P. fluorescens isolated from raw milk. However, Allison et al.

(1998) suggested the quorum sensing systems to be involved in promoting cell

attachment and biofilm formation in P. fluorescens B52, but that do not involved

short chain HSLs.

Figure 10 – Biofilm formation by P. fluorescens wild type (07A and 041) and

transconjugant (07A-2 and 041-3) in minimal medium ABC after

incubation for 48 h at 25 °C, determined as absorvance of crystal violet

at 570 nm.

3.3.8.2. Swarming motility

To test swarming motility, the strains 07A and 041 were point inoculated into

medium containing 0.4% agar. Under the conditions used, only P. fluorescens 07A

was capable of swarming (Figure 11). When AiiA was expressed in P. fluorescens

07A, swarming motility was reduced, indicating that a factor required for swarming

was compromised in this strain or that this assay was compromised due to the

sensibility of this methodology since P. fluorescens 07A transconjugant was grown

previously in LB supplemented with gentamicin.

Figure 11 – Ability to form a swarming colony on ABC after incubation for 18 h at

25 °C. (A) 1, P. fluorescens 07A wild type; 2, P. fluorescens 07A-2

transconjugant. (B) 1, P. fluorescens 041 wild type; 2, P. fluorescens

041-3 transconjugant.

1 2

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

07A 07A-2 O41 041-3

P. fluorescens

Absorbance at 570 nm

3.3.8.3. Extracellular protease

AHL-dependent quorum sensing systems control the production of

extracellular proteolytic activity in many gram-negative bacteria (WHITEHEAD et

al., 2001). To test whether expression of proteolytic activity is generally AHL-

regulated in P. fluorescens 07A and 041, wild type and transconjugant strains were

streaked onto LB agar plates supplemented with 2% skim milk. Clearing zones,

which are indicative of protease activity, were observed for both wild type and

transconjugant strains after 18 h of incubation (Figure 12). Besides, heterologous

expression of AiiA did not decrease proteolytic activity in both strains when they

were grown into different broth media (Figure 13), indicating that the AHL-

dependent regulation of this phenotype is not conserved in these strains of

P. fluorescens isolated from refrigerated raw milk.

Figure 12 – Proteolytic activity on LB agar plates supplemented with skim milk

powder 2% after incubation for 18 h at 25 °C. 1, P. fluorescens 07A

wild type; 2, P. fluorescens 07A transconjugant; 3, P. fluorescens 041

wild type; 4, P. fluorescens 041 transconjugant.

Figure 13 – Proteolytic activity on supernatant of ABC (minimal medium), MMS

(minimal medium) and TYEP medium inoculated with P. fluorescens

wild type (07A and 041) and transconjugants (07A-2 and 041-3) after

24 h of incubation at 25 °C.

3.3.9. Chemical characterization of signal molecules produced by P. fluorescens

07A into TYEP medium

The extracts of signal molecules obtained from TYEP medium were analysed

using mass spectrometry. AHLs molecules were not detected in the samples (Table

6). However, two compounds (4-Dimethylaminobenzaldehyde and Cyclo(ile-val))

were present into the extract obtained from TYEP medium inoculated with P.

fluorescens 07A and absent into TYEP medium not inoculated (Table 6).

A variety of cyclic dipeptides (DKPs) in the signal molecule extract were

obtained from TYEP medium inoculated and not inoculated with P. fluorescens 07A

(Table 6). Prasad (1995) showed that DKPs existed in protein hydrolysates as well as

in fermentation broths and cultures of yeast, fungi, and bacteria. According to

Holden et al. (2000), DKPs were identified as a consequence of their ability to

activate biosensors previously considered specific for AHLs. Although DKPs are

structurally quite distinct, at high concentrations they are able to cross-activate AHL-

dependent reporter constructs based on several different LuxR homologues

(HOLDEN et al., 1999). The detection of these DKPs appears to be an example of

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

07A 07A-2 O41 041-3

P. fluorescens

Proteolytic activity (Units h

-1

µg

-1

)

ABC

MMS

TY EP

fortuitous chemical crosstalk and raises the obvious question as to their origin and

biological function(s). Therefore, although it is quite feasible that the DKPs do not

function as bacteria-to-bacteria signaling molecules per se, they might have a role in

modulating prokaryotic-eukaryotic interactions. In bacteria, the production of DKPs

is not limited to P. aeruginosa, as other gram-negative bacteria, including E. coli and

P. fluorescens (HOLDEN et al., 1999; HOLDEN et al., 2000). According to Cui

(2004), these molecules demonstrated the complexity of quorum sensing and the

existence of cross-communication in signalization systems of P. fluorescens.

Table 6 – Results of chemical characterization of signal molecule extract obtained

from TYEP medium not inoculated and inoculated with P. fluorescens

07A.

Compound

Negative control

TYEP medium

TYEP medium inoculated

with P. fluorescens 07A

Benzaldehyde + -

Caprylic acid + -

Decanoic acid + -

Hexanoic acid + -

Tributylphosphate + +

Quinaldaldehyde + -

1,4-Diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane + +

3-Benzyl-1,4-diaza-2,5-dioxobicyclo[4.3.0]nonane + +

4-Dimethylaminobenzaldehyde* - +

Cyclo(ala-pro) + +

Cyclo(ile-val)* - +

Cyclo(met-pro) + +

Cyclo(pro-leu) + ±

Cyclo(pro-val) + +

*Compound present into TYEP inoculated with P. fluorescens 07A and absent into TYEP not

inoculated. +: present; -: absent; ±: doubt.

3.3.10. Detection of AI-2

The results demonstrated in Table 7 suggest that P. fluorescens isolated from

cooled raw milk produces AI-2 since the sterilized supernatant of these strains

inoculated into LB broth was able to induce bioluminescence production by the

monitor strain V. harveyi BB170.

Table 7 – Detection of auto-inducer two in supernatant of LB broth inoculated with

P. fluorescens. V. harveyi BB170 was used as a monitor strain and V.

harveyi BB120 was used as a positive control.

Strains and medium Luminescence at 175 nm*

P. fluorescens 07A 9766 ± 728

P. fluorescens 041 12567 ± 2145

P. fluorescens 097 9933 ± 634

P. fluorescens 0109 10420 ± 652

V. harveyi BB120 6360 ± 1643

LB medium (negative control) 6776 ± 553

AB medium (negative control) 3111 ± 644

Average and standard deviation of data is shown. n: number of repetitions equal 8.

The occurrence of luxS-dependent AI-2 signaling is widespread among both

gram-negative and gram-positive bacteria including E. coli (pathogenic and non-

pathogenic varieties), Salmonella Typhimurium, Shigella flexneri, Helicobacter

pylori, Streptococcus pyrogenes, Neisseria meningitides, Actinobacillus

actinomycetemcomitans and Porphyromonas gingivalis (SURETTE et al., 1999;

MILLER and BASSLER, 2001). Thus, AI-2 has evolved several diverse, species

specific roles while simultaneously remaining a universal signal recognizable across

numerous species of bacteria. It is clear that AI-2 signaling regulates the expression

of numerous genes and is involved in determining phenotypes, but exactly what AI-2

is signaling is a murky subject (LERAT and MORAN, 2004). Emerging evidence

indicates that AI-2 may not be a density-dependent signal, but rather a waste product.

3.4. CONCLUSIONS

Strains of P. fluorescens isolated from cooled raw milk do not produce AHL

and do not have the genes mupI and phzI. On the other hand, these strains might

produce AI-2.

P. fluorescens 07A and 041 produces less biofilm in LB and MMS than in

ABC minimal medium, and the strain 041 binds better than 07A in polystyrene

microtiter dishes.

Cyclic dipeptides found into the medium induced A. tumefaciens AHL sensor

strains and caused false positive results in the TLC and cross-streak assays.

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CHAPTER 4

QUORUM SENSING IN PSYCHROTROPHIC STRAINS ISOLATED FROM

REFRIGERATED RAW MILK

4.1. INTRODUCTION

Product contamination with psychrotrophic microorganisms is a particular

concern for the dairy industry, as dairy products are maintained and distributed at

temperatures permissive for the growth of these organisms. The diverse

microorganisms categorized as psychrotrophic are ubiquitous in nature and can be

isolated from soil, water, and vegetation (DOGAN and BOOR, 2003).

Gram-negative bacteria usually account for more than 90% of the microbial

population in cold raw milk that has been stored (COUSIN, 1982; PINTO 2004;

MUNSCH-ALATOSSAVA and ALATOSSAVA, 2006). The gram-negative

microbiota is composed mainly of psychrotrophic species of Pseudomonas,

Achromobacter, Aeromonas, Serratia, Alcaligenes, Hafnia, Chromobacterium,

Flavobacterium, and Enterobacter (GARCÍA-ARMESTO and SUTHERLAND,

1997; SØRHAUG and STEPANIAK, 1997; RYSER, 1999; PINTO, 2004). Most of

these bacteria produce extracellular proteolytic and lipolytic enzymes that are

secreted into the milk. Many of these enzymes are not inactivated by pasteurization

or by ultra-high temperature treatment (GRIFFITHS et al., 1981). The residual

activities of these enzymes can reduce the sensorial quality and shelf-life of

processed milk products (FAIRBAIRN and LAW, 1986; DOGAN and BOOR, 2003;

PINTO, 2004; MARTINS et al., 2006).

Among the most common psychrotrophs isolated from raw milk, Aeromonas

hydrophila and Hafnia alvei are considerated opportunistic foodborne pathogens

associated with cases of gastroenteritis. A. hydrophila is a widespread bacterium

found in water, domestic animals, and foods (fish, shellfish, poultry, milk, and raw

meat). This pathogen produces virulence factors, including exotoxins and cytotoxins,

and multiple resistance of the bacterium to many antimicrobials is a fact of high

significance (DASKALOV, 2006).

H. alvei is a gram-negative facultatively anaerobic bacillus that belongs to the

family of Enterobacteriaceae (RODRÍGUEZ et al., 1999). It possesses several

different virulence mechanisms, which are similar or identical to those of other gram-

negative enteropathogens. It is suspected to cause a variety of intestinal disorders and

other illnesses, including pneumonia, meningitis, abscesses, and septicemia

(ALBERT et al., 1992; RODRÍGUEZ et al., 1999).

Another important representative of Enterobacteriaceae isolated from milk is

Enterobacter sp. (COUSIN, 1982; PINTO, 2004). This bacterium produces

extracellular enzymes that can compromise the sensorial quality of dairy products.

Gram et al. (1999) showed that strains of Enterobacteriaceae isolated from

foods produce acyl-homoserine lactones (AHLs) as signal molecules and regulate the

expression of some genes in response to density in a mechanism known as quorum

sensing (QS). According to Gram et al. (1999), the production of signal molecules

was detectable from naturally contaminated foods and from samples to which pure

cultures have been added and the Enterobacteriacea reached 10

to 10

CFU/g. This

high number of bacteria is not uncommon in foods, which indicates that AHLs could

be implicated in regulating phenotypes important in food spoilage and thus possibly

play a role in food quality deterioration.

According to Christensen et al. (2003), several hydrolytic enzymes produced

by a typical member of a food spoilage flora are regulated by QS. These authors

demonstrated that QS is involved in the production of spoilage characteristics in situ

on food products.

Besides the control of spoilage enzyme expression, QS is also related to

expression of virulence genes. A useful host model for studying innate immune

responses to bacterial pathogens is the nematode Caenorhabditis elegans, which

lacks an adaptive immunity (CARDONA et al., 2005). This model is genetically

tractable from the perspectives of both host and pathogen, and thus, serves to

investigate evolutionary conserved mechanisms of microbial pathogenesis and innate

immunity (KURZ et al., 2003; CARDONA et al., 2005).

As QS is related to regulation of several phenotype characteristics, the control

of this mechanism, usually referred to quorum quenching, has been studied and

proved to be successful in microorganisms isolated from non-food sources (DONG et

al., 2000; ULRICH, 2004; WOPPERER et al., 2006). Enzymatic cleavage,

specifically lactone ring hydrolysis, of AHL molecules by numerous Bacillus species

has been reported (DONG et al., 2002). These enzymes, termed lactonases AiiA,

hydrolyze the lactone bond within the AHL moiety, thus changing the relative

conformational structure of the signaling molecule, which prevents binding to the

LuxR transcriptional regulator (ULRICH, 2004). Rasmussen et al. (2005) found that

garlic extract was one of the most effective inhibitors of QS since it reduced

P. aeruginosa biofilm tolerance to tobramycin treatment and virulence of this

bacterium against C. elegans.

The understanding of the role of the QS mechanism in the regulation of

spoilage phenotypes in bacteria from milk is relevant and may be used to create new

strategies to preserve dairy products. Therefore, the purpose of the present work was

to elucidate which signal molecules are produced by proteolytic pychrotrophic

bacteria isolated from cooled raw milk and to relate the QS mechanism to the

spoilage potential and pathogenicity of these strains.

4.2. MATERIAL AND METHODS

4.2.1. Strains and growth conditions

The psychrotrophic strains and other bacteria used in the present study are

listed in Table 1. Unless otherwise stated, these strains were grown at 30 °C in Luria-

Bertani (LB) medium or AB minimal medium (CLARK and MAALOE, 1967)

supplemented with 10 mM glucose (ABG). Vibrio harveyi strains were grown at

30 °C in AB Vibrio medium (BASSLER et al., 1994). Solid media were routinely

solidified with 1.4% agar. Antibiotics were added as required at final concentrations

of 20 µg/ml for gentamicin and tetracycline, 50 µg/ml for trimethoprim and

spectinomycin, 100 µg/ml for ampicillin, and 10 µg/ml for chloramphenicol.

Kanamycin was used at 30 µg/ml for V. harveyi and at 50 µg/ml for E. coli S17-1

and psychrotrophic strains. Besides, tellurite, a selective agent, was added when

required at a final concentration of 100 µg/ml. Growth of liquid cultures was

monitored spectrophotometrically as described in chapter 3, item 3.2.1.

Table 1 - Bacterial strains and plasmids used in this study

Strain Plasmid Description Reference or source

Aeromonas

hydrophila 099

Wild type, psychrotrophic

isolated from cooled raw milk

Pinto, 2004

Agrobacterium

tumefaciens A136

pCF373, pCF218, Tc

, Spc

As described in chapter 3,

item 3.2.1

A. tumefaciens

NTL4

pZLR4, Gm

As described in chapter 3,

item 3.2.1

Burkholderia

cepacia H111

As described in chapter 3,

item 3.2.1

Burkholderia

vietinamensis

As described in chapter 3,

item 3.2.1

Chromobacterium

violaceum CV026

As described in chapter 3,

item 3.2.1

Enterobacter sp.

067

Wild type, psychrotrophic

isolated from cooled raw milk

Martins et al., 2005

Enterobacter sp.

067-7

pBHR1-aiiA-km

Transconjugant, express the

lactonase enzyme

This study

Escherichia coli

HB101

pRK600, Cm

As described in chapter 3,

item 3.2.1

E. coli MT102 pSB403, Tc

As described in chapter 3,

item 3.2.1

E. coli S17-1 pBHR1-aiiA-km

Donor of pBHR1-aiiA, km

that codify the lactonase

enzyme

Ulrich, 2004

E. coli XL1-Blue pMLBAD-aiiA-Trm

-Gm

Donor of pMLBAD-aiiA,

Trm

that codify the

lactonase enzyme

This study

E. coli XL1-Blue pQE30-Xa

As described in chapter 2,

item 2.2.1

This study

E. coli XL1-Blue pQE30-Xa-halI068

Express AHL synthase, HalI,

from H. alvei 068

This study

Hafnia alvei 059

Wild type, psychrotrophic

isolated from cooled raw milk

Martins et al., 2005

H. alvei 068

Wild type, psychrotrophic

isolated from cooled raw milk

Martins et al., 2005

H. alvei 068-1 pBHR1-aiiA-km

Transconjugant, express the

lactonase enzyme

This study

H. alvei 071

Wild type, psychrotrophic

isolated from cooled raw milk

Martins et al., 2005

H. alvei 071-1 pBHR1-aiiA-km

Transconjugant, express the

lactonase enzyme

This study

Pantoea sp. 039

Wild type, psychrotrophic

isolated from cooled raw milk

Martins et al., 2005

Pseudomonas

aeruginosa PAO1

As described in chapter 3,

item 3.2.1

Pseudomonas putida

F117

pAS-C8, Gm

As described in chapter 3,

item 3.2.1

P. putida F117 pKR-C12, Gm

As described in chapter 3,

item 3.2.1

Serratia liquefaciens

MG1

Positive control in the

chitinase assay

Laboratory of

Microbiology,

University of Zürich

Vibrio harveyi

BB120

As described in chapter 3,

item 3.2.1

Vibrio harveyi

BB170

As described in chapter 3,

item 3.2.1

4.2.2. Amplification and sequencing of 16S rDNA from psychrotrophic strains

The identification of psychrotrophic isolates from cooled raw milk was

initially done by Pinto (2004) and in order to confirm their identities API ID32E

(BioMérieux, Marcy-l’Etoile, France) was used for phenotypic characterization.

Thereafter, 16S rDNA was sequenced as described by Juretschko et al. (1998).

DNA manipulations were conduced as described in chapter 2, item 2.2.6.1.

For amplification of the 16S rDNA, the PCR reaction consisted of 25 mM MgCl

5.0 µl of 10X buffer Ex Taq, 25 mM deoxynucleotide triphosphates (dNTPs), 25

pmol of each primer, 1 U Ex Taq DNA polymerase, and 40 ng of DNA in a final

volume of 50 µl. Primers described by Juretschko et al. (1998) (616V

5’AGAGTTTGATYMTGGCTC 3’ and 630R 5’CAKAAAGGAGGTGATCC 3’)

were synthesized by Microsynth (Zürich, Switzerland). PCR reactions were carried

out in a T3 thermocycler (Biometra

, Biolabo Scientific Instruments, Zürich,

Switzerland). The M13 Forward and Reverse primers were used to sequence the

rDNA 16S genes cloned into pCR2.1-TOPO. Thereafter, the obtained sequences

were used to search for similarity using the Ribosomal Database Project II

(http://rdp.cme.msu.edu/seqmatch/seqmatch_result.jsp?qvector=204&depth=0&curre

ntRoot=419&num=20. Access on June 4

, 2007).

4.2.3. Milk spoilage potential and production of exoenzymes by psychrotrophic

strains

To evaluate the milk spoilage potential, samples of reconstituted 12% (w/v)

skim milk powder were inoculated with approximately 1 x 10

CFU/ml of strains

059, 067, 068, 071, or 099. The samples were incubated at 30 °C for 18 h and their

sensorial quality was checked.

Prior to the enzymatic assays, the method of Bradford (BRADFORD, 1976)

was used for quantitative protein determination using bovine serum albumin as a

standard. Proteolytic activity was determined by streaking the strains on LB agar

plates supplemented with 2% (w/v) skim milk powder (WOPPERER et al., 2006)

and on azocasein as described in chapter 2, item 2.2.3.2.

100

Supernatant proteins from the crude extracts of psychrotrophic strains in LB

were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE; LAEMMLI, 1970). After electrophoresis, the gels were stained with

Coomassie brilliant blue. Exoprotease activities of culture supernatants from

psychrotrophic strains were analysed by SDS-PAGE with 0.2% (w/v) azocasein

incorporated into the gel matrix (12% polyacrylamide) as described in chapter 2,

item 2.2.4.

Lipolytic activity was determined by streaking the strains on medium 884

(Tween 80-Agar). Besides, the lipolytic activity on p-nitrophenylpalmitate was

investigated using 100 µl bacterial supernatant from overnight cultures in LB or

TYEP as described in chapter 2, item 2.2.3.3.

Chitinase activity was determined by streaking the strains on ABC minimal

medium supplemented with 2% chitin solution.

4.2.4. Detection and quantification of AHL

AHL production was investigated by cross-streaking psychrotrophic strains

that were grown overnight on LB agar plates against monitor strains as described in

chapter 3, item 3.2.2.

Escherichia coli MT102 pSB403 was used for quantification of

bioluminescence induced by AHL molecules as described in chapter 3, item 3.2.2.

4.2.5. Extraction of AHLs from supernatants

Psychrotrophic strains (10

CFU/ml) were inoculated in 250 ml of LB or 400

ml of AB minimal medium supplemented with 10 mM citrate (ABC medium).

Samples were incubated at 30 °C for 20 h skaking at 300 RPM or until the

population reach 10

CFU/ml. Then, the cells were harvested by centrifugation at

10,000 g for 20 min and cell free supernatants were used to extract AHLs as

described in chapter 3, item 3.2.3.

101

4.2.6. Detection of AHL using Thin Layer Chromatography (TLC)

The extracts were loaded on TLC plates as described in chapter 3, item 3.2.5.

To 150 ml of soft agar at 42 °C, 30 ml of the monitor strain E. coli MT102 (pSB403)

or C. violaceum CV026 was mixed. The soft agar supplemented with the

appropriated monitor strain was dispensed on a TLC plate to produce a 2 to 3 mm

thick layer. After 20 min, the plate was transferred to an airproof box with a wet

paper inside and incubated overnight at 30 °C.

The documentation was dependent on the monitor strain used. For C.

violaceum CV026, the material was incubated up to 48 h and the signal molecules

were identified by formation of violet pigments. When E. coli MT102 pSB403 was

the monitor, the material was incubated overnight at 30 °C, transferred to a dark box

and bioluminescence was detected with a highly sensitive photon-counting camera

(C2400-40; Hamamatsu Photonics Herrsching, Germany).

4.2.7. LC-MS analysis of AHL extracts from bacterial supernatants

One hundred and twenty microliters of dichloromethane extracts from 400 ml

of culture supernatant in ABC or ABG minimal medium were evaporated under a

gentle stream of nitrogen. The residue was re-dissolved in 120 µl of 60% (v/v)

aqueous methanol and separated by reversed-phase LC-MS (C18 column, Grom-Sil

120 ODS-4 HE, 4.6 x 250 mm, Stagroma, Germany) under the following conditions:

a flow rate of 1 ml/min; solvent A UV-treated H

O and 0.1% formic acid; solvent B

acetonitrile (ACN) and 0.1% formic acid. After separation, the mixture was analyzed

by mass spectrometry (LCQ Duo Mass Spectrometer, Thermoquest, Finnigan) using

an electrospray source. The following gradient was applied: solvent B from 25%

ACN to 100% in 20 min, isocratic 5 min.

4.2.8. Resistance of psychrotrophic strains against different antimicrobials

Psychrotrophic strains were inoculated into 5 ml of LB broth containing 100

µg/ml tellurite or antibiotics (100 µg/ml trimethoprim, 20 µg/ml chloramphenicol, 25

102

µg/ml gentamicin, 100 µg/ml ampicillin, 50 µg/ml kanamycin, 20 µg/ml tetracycline,

and 50 µg/ml spectinomycin). Growth at 30 °C was observed at 600 nm after

incubation for 48 h.

4.2.9. Conjugative plasmid transfer and confirmation of identity of

transconjugant strains

Plasmids were delivered to psychrotrophic strains (067, 068 and 071) by

triparental mating as described previously in chapter 3, item 3.2.9. Briefly, donor

(E. coli S17-1 pBHR1-aiiA, km

) and recipient strains, as well as the helper strain E.

coli HB101 (pRK600), were grown overnight in 5 ml of LB medium supplied with

the appropriate antibiotics. After subculturing to an optical density at 600 nm of 0.9,

the cells from 2 ml of culture were harvested, washed, and resuspended in 500 µl of

LB medium. Donor and helper cells (100 µl each) were mixed and incubated for 10

min at room temperature. Then, 200 µl of the recipient cells was added and the

mixture was spot inoculated onto the surfaces of preheated LB agar plates. After

overnight incubation at 30 °C, the cells were plated on LB containing 100 µg/ml

tetracycline and 50 µg/ml kanamycin for counter selection of the donor, helper, and

untransformed recipient cells of 067. Tetracycline at 100 µg/ml and kanamycin at

25 µg/ml were used for counter selection of the donor, helper, and untransformed

recipient cells of 068 and 071.

The transconjugant strains were characterized by restriction digest of pBHR1-

aiiA with NcoI and EcoRI according to Ulrich (2004) and the identity of these strains

was checked using the indol test since E. coli is indol positive and H. alvei and

Enterobacter are indol negative. In addition, the AHL synthase gene halI of the

transconjugant strains was amplified by PCR as described in 4.2.12.

4.2.10. Phenotypic characterization of wild type and transconjugant strains

Proteolytic activity was determined by inoculating the strains into pasteurized

milk, by streaking them on LB agar supplemented with 2% (w/v) skim milk, and

with the azocasein assay as described in chapter 2, item 2.2.3.2.

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4.2.11. Detection and sequencing of a native plasmid from H. alvei 068 and 071

The native plasmid DNA of H. alvei 068 and 071 was isolated using the

QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) and named pMLM. After

isolation, it was loaded on an agarose gel and purified using the QIAquick gel

extraction kit (Qiagen, Hilden, Germany) and digested with restriction enzymes

(ApaI, AvaI, BamHI, BlnI, BsiWI, BsmI, BstBI, BstXI, ClaI, DdeI, EcoRI, EcoRV,

HindIII, KpnI, MluI, MspI, NcoI, NotI, NsiI, NspI, PvuI, RsaI, RpnI, SacI, SalI, SmaI,

SpeI, SphI, StuI, XbaI, XhoI, and XhoII) in order to generate fragments to clone and

sequence.

The fragments obtained were cloned into Zero Blunt

TOPO

PCR Cloning

Kit (Invitrogen, United Kingdom) and the M13 Forward and Reverse primers were

used for sequencing. Thereafter, primers based on the sequences of pMLM068 were

constructed (Table 2) and synthesized by Microsynth (Zürich, Switzerland).

Table 2 - Primers used to sequence pMLM

Primer Sequence (5’-3’)

pMLMA

pMLMB

pMLMC

pMLMCD

CCT ATC CTG CAT CGT GTT

GGT AGC GTA AAA ATT TGC GG

GTA GAG GCA TTT ACG GCG TTT

CAG TGG GTC AGT TCA TGC AA

4.2.12. DNA manipulations, PCR reactions and sequencing of halI and halR

genes

DNA manipulations were developed as described in chapter 2, item 2.2.6.1.

To amplify the AHL synthase gene (halI) and the gene halR that encodes the AHL

receptor (HalR) by PCR, the reaction consisted of 2.0 mM MgCl

, 5.0 µl of 10X

buffer Ex Taq, 2.5 mM deoxynucleotide triphosphates (dNTPs), 25 pmol of each

primer, 1 U Ex Taq DNA polymerase, and 40 ng of DNA in a final volume of 50 µl.

Primers based on the sequences of halI and halR genes (GenBank accession number

AF503776) of H. alvei were constructed (Table 3) and synthesized by Microsynth

104

(Zürich, Switzerland). PCR reactions were carried out in a T3 thermocycler

(Biometra

, Biolabo Scientific Instruments, Zürich, Switzerland).

The M13 Forward and Reverse primers were used to sequence the halI and

halR genes cloned into pCR2.1-TOPO.

Table 3 - Primers used to amplify halI and halR genes by PCR

Primer Sequence (5’-3’) Application

halI-F AACTGATTACACCAATGCAGT Amplification and

sequencing of halI

halI-R GGAATGCTTGAACTATTTGATG Amplification and

sequencing of halI

halI-bam ATTGGATCC

TACACCAATGCAGTCTTAATT Amplification of halI

gene and preparation for

cloning in pQE-30Xa

halI-sac ATTGAGCTC

ATGCTTGAACTATTTGATGTC Amplification of halI

gene and preparation for

cloning in pQE-30Xa

halR-F CTT CAG GGA TGC CAT ATG TTT Amplification and

sequencing of halR

halR-R ACT GCA TTG GTG TAA TCA GTT Amplification and

sequencing of halR

The introduced restriction sites for BamHI and SacI are underlined.

4.2.13. Cloning and heterologous expression of AHL synthase (halI) of H. alvei

068 in pQE-30Xa

Once the complete sequence of the halI gene was obtained, primers (Table 3)

were designed to amplify the open reading frame (ORF) by PCR using the bacterial

genomic DNA as a template and TaKaRa Ex Taq polymerase. Primers generated

BamHI and SacI sites at the 5’ and 3’ ends of the amplificates, respectively. The

DNA amplificate, 660 bp, containing the halI structural gene, was digested with

BamHI and SacI and ligated into vector pQE-30Xa (Qiagen) cut with the same

restriction enzymes. A plasmid harbouring the ORF of halI inserted downstream of

the T5 promoter was selected and named pQE-30Xa-halI068. The plasmid was

transformed into the expression strain E. coli XL1-Blue.

For overproduction of HalI, E. coli XL1-Blue cells carrying pQE-30Xa-

halI068 was grown in dYT medium as describe previously in chapter 2, item 2.2.7.

At an optical density at 600 nm of 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG)

105

was added to the culture to a final concentration of 1 mM. After 5 h incubation at 37

°C, the cells were collected by centrifugation at 10,000 g for 30 min and resuspended

in 50 mM Tris-HCl (pH 8.0). Then, 3 µl of cell suspension was loaded on SDS-

PAGE (15%) to detect HalI.

4.2.14. Detection, extraction, and characterization of AHLs encoded by halI

AHL production was investigated by cross-streaking E. coli XL1-Blue pQE-

30Xa-halI068 cells that were grown overnight on dYT agar plates supplemented with

1 mM IPTG against E. coli pSB403 or C. violaceum CV026, as described in chapter

3, item 3.2.2.

To extract AHLs, 10

CFU/ml of E. coli XL1-Blue pQE-30Xa-halI068 were

inoculated into 250 ml of dYT or in 400 ml of AB minimal medium supplemented

with 15 mM glucose (ABG). At an optical density at 600 nm of 0.5, IPTG was added

to the culture to a final concentration of 1 mM. The samples were incubated at 30 °C

up to 48 h with skaking at 350 RPM. Then, the cells were harvested by centrifugation

at 10,000 g for 20 min and cell free supernatants were used to extract AHLs as

described in chapter 3, item 3.2.3.

The extract was loaded on TLC plates drop by drop of 2 µl and the TLC was

dried in cold air step by step as described in chapter 3, item 3.2.5. Aliquots of 150 ml

of soft agar at 42 °C were mixed with 30 ml of the monitor strain E. coli MT102

pSB403 or C. violaceum CV026. The soft agar supplemented with the appropriated

monitor strain was dispensed on a TLC plate to produce a 2 to 3 mm thick layer.

After 20 min, the plate was put in an airproof box with a wet paper inside and

incubated overnight at 30 °C. The documentation was developed as described in

chapter 4, item 4.2.6.

Chemical characterization of AHL molecules encoded by halI was performed

in extracts from bacterial supernatants obtained from AB minimal medium

supplemented with 15 mM glucose by LC-MS as described in chapter 4, item 4.2.7.

106

4.2.15. Detection of AI-2 in supernatant of LB medium inoculated with

psychrotrophic strains

Psychrotrophic strains were grown overnight with aeration at 30 °C on LB

medium. The autoinducer two was detected as described in chapter 3, item 3.2.12.

4.2.16. Pathogenesis of psychrotrophic strains against Caenorhabditis elegans

4.2.16.1. Maintenance and cultivation of

C. elegans

C. elegans was sustained on NGM I plates covered with E. coli OP50 untill

three weeks at 20 °C. Plates of NGM I were inoculated with 100 µl of a fresh

overnight culture of E. coli OP50 and incubated at 37 °C overnight. Then, overgrown

plates were stored at 4 °C. During the assays, the nematodes were transferred every

two days onto fresh E. coli plates. For this, a piece of agar covered with E. coli and

C. elegans was cut with a sterile scalpel and put onto a new E. coli plate.

4.2.16.2. Egg preparation of

C. elegans

To synchronize all C. elegans at the same developmental stage, plates with

plenty of eggs were used. Worms and eggs were rinsed from plates four times with

1 ml of sterile water and the suspension was dispersed in three tubes of 2 ml. The

suspension was mixed with 500 µl of 12% sodiumhypochlorite solution and mixed

by vortexing for approximately 8 min or until all worms had dissolved. The

suspension was centrifuged for 1 min at 4 °C and 3,200 RPM. The supernatant was

carefully discarded and the pellet was washed with 1 ml of sterile water. Then, it was

centrifuged for 1 min and the supernatant was discarded. The pellet of tube one was

resuspended with 100 to 200 µl of M9 buffer and the suspension was used to

resuspend the pellets of the other tubes. Thereafter, this solution was transferred to a

NGM I plate with E. coli and incubated at 20 °C.

107

4.2.16.3. Nematode assays

Assays of pathogenesis against C. elegans were performed essentially as

described by Wopperer et al. (2006). Briefly, 100 µl of the suspensions of overnight

cultures of psychrotrophic strains were plated on six-well plates containing nematode

growth medium (NGM II) for slow killing assays. After 24 h of incubation at 30 °C,

a bacterial lawn was formed and approximately 25 hypochlorite-synchronized L4

larvae of Caenorhabditis elegans Bristol N2 (obtained from the Caenorhabditis

Genetics Centre, University of Minnesota, Minneapolis, MN) were used to inoculate

the plates. The actual number of worms was determined by using a Stemi SV6

microscope (Zeiss, Oberkochen, Germany) at a magnification of 50X. Plates were

then incubated at 20 °C and scored for live worms; nematodes were considered dead

when they failed to respond to touch. The percentage of live worms and their

morphological appearance were registered after two days. After five days, the total

number of nematodes, including parental and progeny nematodes, if present, was

scored. All experiments were carried out five times, and E. coli OP50 was used as a

negative control in the assays. A psychrotrophic strain was considered to be

pathogenic for C. elegans if one of the following criteria described previously by

Cardona et al. (2005), and Wopperer et al. (2006) was met: (i) a sick appearance at

day two, including reduced locomotive capacity and the presence of a distended

intestine; (ii) percentage of live worms after two days of ≤ 50%; and (iii) total

number of worms after five days of ≤ 100. For differentiating mild from severe

infections, the presence of one, two, or three of these criteria was scored as 1, 2, or 3,

respectively. A strain was considered pathogenic when at least one criterion was

observed. A strain was described as nonpathogenic when no symptoms of disease

were observed during the course of the infection experiment (pathogenicity score 0).

108

4.3. RESULTS AND DISCUSSION

4.3.1. Confirmation of identity of psychrotrophic strains isolated from cooled

raw milk

The strains used in this work were previously identified by Pinto (2004) as

described in Table 4. In order to confirm the identity of these strains, API ID32E was

used and the obtained results were different from the data described by Pinto (2004)

(Table 4). Therefore, it was necessary to use an accurate assay in order to identify the

strains since both identifications were used phenotypic methods that may give false

positive results. Therefore, the 16S rDNA was sequenced since it is universally

distributed and highly conserved among all organisms and it is known as a gold

standard for discerning evolutionary relationships among prokaryotes (ZHANG et

al., 2002).

Analysis of 16S rRNA gene sequencing and biochemical tests were used by

Janda et al. (2005) to separate Hafnia into different groups. Based upon 16S rRNA

gene sequencing results, two genetic groups were identified and the biochemical test

of malonate utilization was found to be the most differential. The test results of the

malonate utilization assay alone correctly assigned 90% of Hafnia isolates to their

correct DNA group (JANDA et al., 2005).

109

Table 4 – Identification of psychrotrophic strains isolated from cooled raw milk

Strain API 20NE * API ID32E rDNA 16S

039 Serratia liquefaciens Nd** Pantoea sp.

059 Hafnia alvei Nd Hafnia alvei

067 Hafnia alvei Enterobacter cloacae Enterobacter sp.

068 Hafnia alvei Hafnia alvei Hafnia alvei

071 Serratia odorifera Hafnia alvei Hafnia alvei

099 Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila

* Determined by Pinto (2004).

** Nd: not determined.

According to the results obtained, the strains 039 and 067 need further

investigations of their identities since the sequencing of 16S rDNA did not achieve

conclusive results about the identity of these species. A polyphasic approach should

be used to identify these strains in the future.

4.3.2. Spoilage potential and production of exoenzymes by psychrotrophic

strains isolated from cooled raw milk

4.3.2.1. Potential to spoil milk samples

Bacterial spoilage causes significant economic losses for the dairy industry

(COUSIN, 1982; DATTA and DEETH, 2001) and different psychrotrophic strains

can show different spoilage potentials (WIEDMANN et al., 2000; DOGAN and

BOOR, 2003; PINTO, 2004). The strains evaluated in this study showed different

abilities to spoil milk samples: A. hydrophila 099 was the most deteriorative,

whereas H. alvei 059 had the least ability to spoil milk (Figure 1).

The proteolytic activity of some extracellular enzymes of A. hydrophila was

recognized and these enzymes are considered to play a major role in the virulence

and pathogenicity of the bacterium (MEDINA-MARTÍNEZ et al., 2006). Besides,

Vivas et al. (2004) showed that this bacterium can produce and secrete proteases able

to cleave milk proteins. According to Cousin (1982), proteases produced by

Aeromonas are able to degrade α-, β-, κ-, and γ-casein, as well as the whey proteins.

H. alvei strains 059, 068 and 071 presented different spoilage potentials

(Figure 1). According to Bruhn et al. (2004), this bacterium was the dominant

110

member of the Enterobacteriaceae in vacuum-packed meat and it may induce food

quality-relevant phenotypes in other bacterial species in the same environment. Then,

it may influence the spoilage of food products in which Enterobacteriaceae

participate in the spoilage process.

Although Enterobacter sp. is normally isolated from raw and pasteurized

milk and butter, this bacterium is not a potent bacterium for spoilage of dairy

products (COUSIN, 1982); however, in this study, it was verified that Enterobacter

sp. 067 had a large potential to spoil samples of reconstituted skim milk powder

(Figure 1).

Figure 1 – Spoilage ability of psychrotrophic strains inoculated in reconstituted skim

milk powder 12% (w/v) after incubation for 18 h at 30 °C. (C) Negative

control, milk sample not inoculated, (059) H. alvei, (067) Enterobacter

sp., (068) H. alvei, (071) H. alvei, (099) A. hydrophila.

059 067

068

071

099

111

4.3.2.2. Production of extracellular enzymes

a) Protease

Psychrotrophic strains were streaked on LB agar plates supplemented with

2% skim milk powder and it was verified that they had different abilities to produce

proteolytic enzymes able to cleave casein (Figure 2). These results confirmed the

data obtained in the evaluation of spoilage potential and showed that A. hydrophila

produced a high amount of exoproteases compared to the other strains (Figure 2).

Figure 2 – Proteolytic activity on LB agar supplemented with 2% (w/v) skim milk

powder after incubation for 24 h at 30 °C. Clearing zones are indicative

of protease activity. (059) H. alvei, (067) Enterobacter sp, (068) H. alvei,

(071) H. alvei, (099) A. hydrophila.

Proteolytic activity was not detected in the supernatants of LB and TYEP

broth media cultured with strains 039, 059, 067, 068, and 071. However, the strain

099 showed a proteolytic activity of 0.131 units/h/µg protein in TYEP. Viana (2006)

also did not detect proteolytic activity in supernatants of TYEP medium inoculated

with 067 and 071 when it was used the azocasein assay. Maybe the azocasein is not

the best substrate for the determination of proteolytic activity produced by these

strains.

The presence of extracellular proteins was not observed in the supernatants of

LB medium inoculated with 059, 068, and 071 by SDS-PAGE (Figure 3A). Small

extracellular proteins were detected in the supernatants obtained from 039 and 067

(Figure 3A). Although the strains 067, 068, and 071 were proteolytic in LB

supplemented with skim milk powder (Figure 2), proteolytic activity on zymogram

was not detected (Figure 3B). Maybe these enzymes were in low concentration,

059

067

068

071 099

112

azocasein was not the best substrate for them, or they were not renaturated after the

development of the zymogram.

On the other hand, many extracellular enzymes were detected in the

supernatant obtained from A. hydrophila 099 (Figure 3A) and two of them had

proteolytic activity on SDS-PAGE supplemented with 2% azocasein (Figure 3B).

Production of both serine- and metalloprotease activities in A. hydrophila is under

the control of quorum sensing mechanism (SWIFT et al., 1999), which also occurs

for protease production in Aeromonas salmonicida (SWIFT et al., 1999) and other

bacterial pathogens (ZHU et al., 2003).

Figure 3 - SDS-PAGE (A) and zymogram azocasein (B) gels (12%) showing

protease production by psychrotrophic strains after growth in LB

medium for 18 h at 30 °C. Lanes: (S) molar mass standards (BioRad),

(039) Pantoea sp (059), H. alvei, (067) Enterobacter sp., (068) H. alvei,

(071) H. alvei, (099) A. hydrophila.

b) Lipase

Lipolytic activity of gram-negative glucose fermenting isolates from cooled

raw milk was generally associated with lecithinase activity (PINTO, 2004). In this

study, lipolytic activity was not detected in the supernatants of LB and TYEP media

S 039 059 067 068 071 099

250

150

100

150

S 039 059 067 068 071 099

250

100

113

after growth of strains 039, 059, 067, 068, and 071 overnight. Only the strain 099

showed a lipolytic activity of 1.104 units/h/µg protein in TYEP. This activity was

confirmed on Tween 80-Agar (Figure 4). According to Brumlik and Buckley (1996),

among extracellular enzymes released by A. hydrophila, a glycerophospholipid-

cholesterol acyltransferase (GCAT) has been described and characterized. This

enzyme is analogous to the important mammalian plasma enzyme lecithin-

cholesterol acyltransferase and, like most of the lipases found in the microbial world,

is a member of the lipase superfamily (ANGUITA et al., 1993).

Figure 4 – Lipolytic activity after growth of A. hydrophila 099 on Tween 80-Agar for

48 h at 30 °C. Precipitation zones are indicative of lipase activity.

c) Chitinase

Only A. hydrophila 099 showed chitinase activity in ABG minimal medium

supplemented with 2% chitin solution (Figure 5). According to Chen et al. (1991), A.

hydrophila JP101 is able to use chitin as its carbon and nitrogen sources when grown

on chitin medium and apparently it synthesizes the entire enzymatic system through

which degradation of chitin occurs.

Chitinases cleave the β-1,4-glycosidic bonds of chitin, a β-1,4-linked,

unbranched polymer of N-acetylglucosamine, which is a major component of insect

exoskeletons, shells of crustaceans, and fungal cell walls. These enzymes have been

detected in a variety of organisms, including organisms that do not contain chitin as a

structural component, such as bacteria, plants, and animals. Bacteria utilize

099

114

chitinases for assimilation of chitin as a carbon and nitrogen source and these

enzymes play an important ecological role in the degradation of chitin (WU et al.,

2001).

Figure 5 – Chitinase activity after grouth for 5 days at 30 °C on ABG minimal

medium supplemented with 2% (w/v) chitin solution. Clearing zones

are indicative of chitinase activity. (MG1) Positive control: S.

liquefaciens MG1, (059) H. alvei, (067) Enterobacter sp., (068) H.

alvei, (071) H. alvei, (099) A. hydrophila.

4.3.3. Detection of AHL molecules produced by Enterobacter sp., H. alvei, and A.

hydrophila

Psychrotrophic proteolytic strains induced the biosensor strains E. coli

MT102 pSB403, C. violaceum CV026, P. putida F117 pAS-C8, P. putida

F117 pKR-C12, A. tumefaciens NTL4, and A. tumefaciens A136 (Table 5). Only

Pantoea sp. 039 was not able to induce the biosensors. As we used a range of

different AHL monitor systems in combination, it is possible that the entire range of

known AHLs was detected. Members of Enterobacteriaceae isolated from foods

have been shown to produce and secrete signal molecules, such as AHLs (GRAM et

al., 1999; RAVN et al., 2001; GRAM et al., 2002; CHRISTENSEN et al., 2003;

FLODGAARD et al., 2003; BRUHN et al., 2004).

Strains 059, 068, and 071 of H. alvei produced more AHLs than the others,

since they were able to strongly induce the monitor strains (Table 5). Many authors

reported that AHL is ordinarily produced by spoilage bacteria isolated from foods

MG1

059

067

099

068 071

115

(GRAM et al., 1999; RAVN et al., 2001; BRUHN et al., 2004). Pinto et al. (2007)

demonstrated that AHL production is common among psychrotrophic bacteria

isolated from milk and indicated that quorum sensing may play an important role in

the spoilage of this product.

Table 5 – Activation of the AHL monitor strains in cross-streak experiments

Result obtained with Bacteria

CV 026 pSB403 F117

(pAS-C8)

F117

(pKR-C12)

A 136 NTL4

Pantoea sp 039 - - - - - -

H. alvei 059 +++ +++ ++ - +++ +++

Enterobacter sp 067 + ++ - - + ++

H. alvei 068 +++ +++ ++ - +++ +++

H. alvei 071 +++ +++ + - +++ +++

A. hydrophila 099 ++ ++ + - + +++

B. cepacia H111 Nd +++ +++ Nd Nd Nd

B. vietinamensis Nd Nd Nd +++ Nd Nd

P. aeruginosa PAO1 +++ Nd Nd Nd +++ +++

The six monitor strains were cross-streaked against different psychrotrophic strains on LB agar plates.

Following up to 48 hours of incubation at 30

C, the production of violacein by C. violaceum CV026,

bioluminescence by E. coli pSB403, green fluorescent protein gfp(ASV) by P. putida F117, and β-

galactosidase activity by A. tumefaciens A136 and NTL4 was visualized as described in material and

methods. Levels of activation are indicated as follows: +++, strong activation, diffusion of AHL > 1

cm; ++, activation, diffusion of AHL of 0.5 to 1 cm; +, weak activation, diffusion of AHL < 0.5 cm; -,

no detectable activation. Nd: not determined.

4.3.4. Characterization of AHL molecules using TLC

Enterobacter sp. 067 and A. hydrophila 099 produced less AHL compared to

H. alvei 059, 068, and 071, since it was necessary to load high volumes of AHL

extracts on TLC plates to detect production of bioluminescence by E. coli pSB403

(Figure 6). Besides, the extracts obtained from Enterobacter sp. 067 were not able to

induce C. violaceum CV026 (Figure 7), which indicated that it does not produce

C4-HSL. These results confirmed the data obtained in the cross-streak experiments

and indicated that these bacteria produce different AHL molecules.

AHL was not detected on the TLCs loaded with supernatant of LB inoculated

with Pantoea sp. 039 (Figure 6B and 7B). However, Pantoea stewartii subsp.

stewartii, a

bacterial pathogen of sweet corn and maize, used quorum sensing to

116

control the extracellular

polysaccharide stewartan, a major virulence factor of this

strain (BODMAN et al., 1998).

Figure 6 - A representative thin-layer chromatograms of the signal molecules present

in cell free supernatants of Enterobacteriaceae isolated from cooled raw

milk. The spots were detected with the E. coli pSB403 reporter strain.

Standards: N-(hexanoyl)-DL-homoserine lactone (HHL); N-(octanoyl)-L-

homoserine lactone (OHL); N-(dodecanoyl)-L-homoserine lactone

(DHL); N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). (059) H.

alvei, (067) Enterobacter sp., (068) H. alvei, (039) Pantoea sp., (071) H.

alvei, (099) A. hydrophila.

Figure 7 - A representative thin-layer chromatograms of the signal molecules present

in cell free supernatants of Enterobacteriaceae isolated from cooled raw

milk. The spots were detected with the C. violaceum CV026 reporter

strain. Standards: N-(3-oxohexanoyl)-L-homoserine lactone (OHHL); N-

(butanoyl)-L-homoserine lactone (BHL); N-(octanoyl)-L-homoserine

lactone (OHL); N-(hexanoyl)-DL-homoserine lactone (HHL). (059) H.

alvei, (067) Enterobacter sp., (068) H. alvei, (039) Pantoea sp., (099) A.

hydrophila, (071) H. alvei.

HHL/OHL DHL 059 067 068

LB medium

1 µl 20 µl 1 µl

OHHL HHL/OHL 039 071

LB medium

30 µl 1 µl

HHL

/OHL

DHL 099

LB medium

12 µl

OHHL BHL HHL/OHL 059 067 068

LB medium

1 µl 30µl 1 µl

OHHL BHL HHL/OHL 039 099 071

LB medium

30 µl 10 µl 1 µl

117

Degradation products of N-(dodecanoyl)-L-homoserine lactone (DHL) were

detected on TLC plates (Figure 6A and C). This suggested high sensitivity of the

AHL molecule to manipulation and exposure to room temperature.

C. violaceum CV026 was unable to detect N-(3-oxohexanoyl)-L-homoserine

lactone (OHHL) (Figure 7). Then, it is important to use multiple AHL sensor systems

to detect a broad range of AHL molecules.

4.3.5. Characterization of AHL molecules by liquid chromatography-mass

spectrometry (LC-MS)

AHL extracts obtained from cultures of psychrotrophic bacteria were

analyzed by liquid chromatography and then pressed through a metal capillary at

high potential in a positive-electrospray ionization high-resolution mass

spectrometer. A mix of standards was used in order to determine the retention time of

each compound and the light ions arrived at the detector sooner than the heavy ones

(Figures 8 and 9).

AHL extracts from Enterobacter sp. 067, H. alvei 068, H. alvei 071, and A.

hydrophila 099 were characterized by MS after calibration of the equipment as

shown in Figure 8. For H. alvei 059, the AHL extract was also characterized by MS

after recalibration of the equipment as shown in Figure 9.

LC-MS of dichloromethane extracts from 400 ml of culture supernatant in

ABG minimal medium of psychrotrophic strains unambiguously detected different

acyl-homoserine lactones with correct retention times. They were further validated

by the accurate masses of the ions, which did not deviate more than 0.015 Da

(Figures 10, 12, 13, and 14). Strains 059, 068, 071, and 099 produced different AHL

molecules. However, it was not possible to detect AHL molecules in the AHL extract

from Enterobacter sp. 067 inoculated in ABG minimal medium (Figure 11),

although it induced the biosensors in the cross-streak assay and on the TLC plates.

118

Figure 8 – High-performance liquid chromatography-positive electrospray ionization

(ESI

)-MS chromatogram showing the mass spectra for the standards: N-

(butanoyl)-L-homoserine lactone (C4-HSL), N-(3-oxohexanoyl)-L-

homoserine lactone (3-oxo-C6-HSL), N-(hexanoyl)-L-homoserine lactone

(C6-HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), N-

(octanoyl)-L-homoserine lactone (C8-HSL), and N-(decanoyl)-L-

homoserine lactone (C10-HSL).

RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Tim e (min)

100

Relative Abundance

15.28

18.61

12.46

10.89

5.46

20.47

20.68

17.28

14.83

21.65

23.13

6.63

23.64

3.56

10.36

3.80

7.70

0.91

1.69

2.36

NL: 1.24E8

m/z=

171.5-172.5+

199.5-200.5+

213.5-214.5+

227.5-228.5+

241.5-242.5+

255.5-256.5 MS

StandardMix_0906_01

C4-HSL

[

M+H

]

= 172

3-oxo-C6-HSL

[

M+H

]

= 214

C6-HSL

[

M+H

]

= 200

3-oxo-C8-HSL

[M+H]

= 242

C8-HSL

[

M+H

]

= 228

C10-HSL

[

M+H

]

= 256

119

Figure 9 - High-performance liquid chromatography-positive electrospray ionization

(ESI

)-MS chromatogram showing the mass spectra for the standards: N-

(butanoyl)-L-homoserine lactone (C4-HSL), N-(hexanoyl)-L-homoserine

lactone (C6-HSL), N-(octanoyl)-L-homoserine lactone (C8-HSL), and N-

(decanoyl)-L-homoserine lactone (C10-HSL).

RT: 0.00 - 19.85

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Tim e (m in)

100

Relative Abundance

14.26

9.69

4.29

15.40

15.95

17.59

10.59

0.57 0.98 1.92 2.61

5.13 17.96

3.39 11.17

5.45

13.37

6.39

9.258.45

NL: 1.13E8

m/z=

171.5-172.5+

199.5-200.5+

227.5-228.5+

255.5-256.5 MS

StandardMix_0808

_01

C4-HSL

[M+H]

= 172

C6-HSL

[M+H]

= 200

C8-HSL

[M+H]

= 228

C10-HSL

[M+H]

= 256

120

Figure 10 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of H. alvei 059.

Signal molecule extract was obtained from cell-free culture

supernatant in ABG minimal medium. The upper panel shows the

base peaks (total ion current chromatogram). The lower panel shows

the extracted single chromatogram. The retention times as well as

the extracted ion chromatograms for the [M+H]

adducts of four

detected homoserine lactones are shown.

200

242

214

228 at 14.24

RT:

0.00 - 21.01

0 2 4 6 8 10 12 14 16 18 20

Time (min)

100

Relative Abundance

20.58

4.55

12.85

20.42

8.79

15.69

2.85

9.45

20.15

11.61

15.95

14.069.90

17.56

3.63

6.98

8.38

15.485.75

2.07

18.59

1.80

1.11

4.55

20.59

9.45

11.61

14.07

5.75

6.21

12.39

11.18

16.94

16.82

8.12

6.79

1.97 1.57

17.51 0.41 3.65

2.83 18.44

20.00

NL: 1.55E8

Base Peak MS

BG_059_0808

NL:

m/z=

199.5-200.5+

213.5-214.5+

227.5-228.5+

241.5-242.5

059

0808

Relative Abundance

121

RT: 0.00 - 25.01

0 2 4 6 8 10 12 14 16 18 20 22 24

Tim e (m in)

100

Relative Abundance

21.79

240.6

14.05

150.1

21.85

240.6

16.05

214.2

24.26

458.3

19.99

214.2

17.71

214.2

23.29

214.2

18.08

214.2

11.35

136.0

7.89

251.0

10.52

243.2

13.13

237.0

10.34

309.0

4.92

122.2

7.24

316.1

8.49

133.1

0.96

266.9

3.40

104.6

1.67

267.0

NL:

2.46E7

Bas e Peak

ABG_067_

0706_01

Figure 11 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of Enterobacter

sp. 067. Signal molecule extract was obtained from cell-free culture

supernatant in ABG minimal medium.

122

Figure 12 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of H. alvei 068.

Signal molecules extract was obtained from cell-free culture

supernatant in ABG minimal medium. The upper panel shows the

base peaks (total ion current chromatogram). The lower panel shows

the extracted single chromatogram. The retention times as well as

the extracted ion chromatograms for the [M+H]

adducts of three

identified and two questionable homoserine lactones are shown.

188??

214

200

242

300??

Relative Abundance

RT: 0.00 - 25.00

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

100

Relative Abundance

21.89

21.77

21.69

6.47

21.64

14.13

21.47

18.16

21.16

22.66

4.60

16.8516.23

5.07

24.27

20.30

20.01

8.15

10.71

11.68

10.56

14.60

12.464.40

0.11 2.34

6.47

21.84

17.15

4.60

16.23

20.31

23.41

20.08

10.88

8.08

12.46

15.74

4.41

8.53

15.04

9.16

0.67

2.07 3.59

NL: 1.91E8

Base Peak MS

ABG_068_0706_01

NL: 1.41E8

m/z=

187.5-188.5+

199.5-200.5+

213.5-214.5+

241.5-242.5+

299.5-300.5 MS

ABG_068_0706_01

123

Figure 13 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of H. alvei 071.

Signal molecule extract was obtained from cell-free culture

supernatant in ABG minimal medium. The upper panel shows the

base peaks (total ion current chromatogram). The lower panel shows

the extracted single chromatogram. The retention times as well as

the extracted ion chromatograms for the [M+H]

adducts of three

identified and one questionable homoserine lactones are shown.

214

200

242

300?

RT:

0.51 -

2 4 6 8 10 12 14 16 18 20

Time (min)

100

Relative Abundance

14.22

6.64

3.84

21.16

18.22

16.28

16.74

7.72

6.30

18.72

10.79

16.90

13.9511.92

20.08

5.89

8.22

13.23

9.864.73

0.86

1.93 2.75

6.64

17.22

16.28

20.04

18.20

11.04

9.83

15.70

8.20

12.57

3.72

8.653.93 15.14

4.71

3.632.29 1.06

NL: 9.21E7

Base Peak MS

ABG_071_0706_01

NL: 7.23E7

m/z=

199.5-200.5+

213.5-214.5+

227.5-228.5+

241.5-242.5+

299.5-300.5 MS

ABG_071_0706_01

Relative Abundance

124

Figure 14 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of A.

hydrophila 099. Signal molecule extract was obtained from cell-free

culture supernatant in ABG minimal medium. The upper panel

shows the base peaks (total ion current chromatogram). The lower

panel shows the extracted single chromatogram. The retention times

as well as the extracted ion chromatograms for the [M+H]

adducts

of one identified and one questionable homoserine lactones are

shown.

172

186?

RT: 0.00 -

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (min)

100

Relative Abundance

14.27

11.21

11.38

12.10

21.61

12.41

5.84

15.92

24.04

23.05

20.48

19.81

3.67

19.19

17.37

0.76

7.85

1.34

5.02

8.68

7.47

2.31

5.84

7.85

8.68

17.10

21.57

17.98

9.21

10.02

4.78

3.67 0.87

10.95

0.95 21.95

3.47 12.85

16.5013.18

14.74

23.01 18.64

19.78

NL:

8.77E7

Base Peak

BG_099_070

6_01

1.74E

m/z=

171.5-172.5+

185.5-186.5+

199.5-200.5

BG_099_070

6_01

Relative Abundance

125

The results obtained by LC-MS (Figures 8 - 14) are summarized in Table 6.

H. alvei strains 059, 068, and 071 were able to produce 3-oxo-C6-HSL, C6-HSL, and

3-oxo-C8-HSL, whereas C8-HSL was produced by H. alvei 059 and 071 (Table 6).

In the experimental conditions adopted, 3-oxo-C6-HSL was the main AHL

produced by H. alvei strains (Figures 10, 12, 13). This result is in agreement with

Bruhn et al. (2004), who verified that this same HSL was predominant among the

four AHLs produced by H. alvei isolated from vacuum-packed meat.

A. hydrophila 099 was able to produce C4-HSL and C6-HSL in ABG

minimal medium (Figure 14, Table 6), which is in agreement with Swift et al.

(1997), who demonstrated that A. hydrophila produces C4-HSL as the principal AHL

molecule. A quorum sensing system in this pathogen has been associated with the

regulation of biofilm development (LYNCH et al., 2002) and exoprotease production

(SWIFT et al., 1999). Medina-Martínez et al. (2006) showed an effect of

environmental conditions, such as temperature and glucose concentration, on

C4-HSL production by A. hydrophila and this warrants further investigation to

elucidate the effect of external conditions on the production of AHL signal molecules

to reveal the relevance of quorum sensing in food storage.

H. alvei produced a molecule that presented a mass spectrum similar to

3-hydroxy-C4-HSL and 3-hydroxy-C12-HSL, whereas A. hydrophila 099 probably

produced C5-HSL (Table 6). However, the identity of these molecules was not

confirmed, since standards for these compounds were not available to determine their

mass spectrums and retention times.

Characterization of different AHLs reinforce the data obtained in the cross-

streak assay when the induction of different biosensor strains by H. alvei was

verified. Each AHL biosensor relies on a particular LuxR family protein, thus

displays specificity towards the cognate AHL, and in some cases, to closely related

AHLs.

126

Table 6 – Summary of identification by high-performance liquid chromatography

positive electrospray ionization (ESI

)-MS of AHLs produced by H. alvei

059, 068, and 071, Enterobacter sp. 067, and A. hydrophila 099

Acyl-homoserine lactones (AHLs)

Isolates

C4-

HSL

3-hydroxi-

C4-HSL

C5-

HSL

3-oxo-

C6-HSL

C6-

HSL

3-oxo-

C8-HSL

C8-

HSL

3-hydroxi-

C12-HSL

H. alvei 059 - - - +

+ + -

Enterobacter sp. 067 - - - - - - - -

H. alvei 068 -

H. alvei 071 - - - +

+ +

A. hydrophila 099 + -

- + - - -

- Absent; + Present; ± doubt.

4.3.6. Resistance of psychrotrophic strains against antibiotics and tellurite

The high incidence of antibiotic resistance detected among psychrotrophic

isolates (Table 7) may demonstrate high selective pressure in raw milk. This datum

was confirmed by Araújo (1998), who found that 95% of 201 strains of

Staphylococcus aureus isolated from raw milk were resistent to different antibiotics,

and by Carneiro and Júnior (2006), who demonstrated that Aeromonas spp. was

resistant to several antimicrobials present in pasteurized milk in Brazil.

Table 7 – Tellurite and antibiotic susceptibility of psychrotrophic strains in LB broth

Strain

Antibiotic

039 059 067 068 071 099

Tellurite - - + + + -

Trimethoprim + + + + + +

Chloramphenicol + + + + + +

Gentamicin - - - - - -

Ampicillin + + + + + +

Kanamycin - - - - - -

Tetracycline + + + + + +

Spectinomycin + - + - - -

Growth is indicated as follows: + growth; - no detectable growth.

127

4.3.7. Quorum quenching mechanism in Enterobacter and H. alvei

4.3.7.1. Mobilization of pBHR1-aiiA

E. coli XL1-Blue pMLBAD-aiiA-Trm

-Gm

was used as the donor in the

triparental mating assay, but since the transconjugant strains did not grow well in LB

supplemented with gentamicin E. coli S17-1 pBHR1-aiiA was used as the donor in

the triparental mating since it carries a plasmid that confers resistance against

kanamycin and the wild type strains were not able to grow in LB supplemented with

this antibiotic.

The plasmid pBHR1-aiiA-Km

was successfully transferred to Enterobacter

sp. 067 (Figure 15). However, it was not possible to detect the aiiA gene after

extraction and digestion of pBHR1-aiiA-Km

from H. alvei 068 and 071

transconjugants (Figure 16). When pBHR1-aiiA-Km

obtained from these strains was

digested with EcoRI and NcoI, some additional bands on the agarose gel were

verified (Figure 16) and one of these bands was similar to a native plasmid that

contains a high number of copies.

Figure 15 – Agarose gel electrophoresis of restriction reactions of pBHR1-aiiA-Km

containing the cloned aiiA gene. Lane 1, Standard; lanes 2, 3, 4, 5, 6,

and 7, 067 transconjugants; lane 8, positive control: E. coli XL1-Blue

pBHR1-aiiA-Km.

2000

250

750

500

10000

1 2 3 4 5 6 7 8

1500

pBHR1-Km

aiiA

1000

128

Figure 16 – Agarose gel electrophoresis of restriction reactions of pBHR1-aiiA-Km

containing the cloned aiiA gene. Lanes 1 and 7, standards; lanes 2 and

3, 068 transconjugants; lanes 4 and 5, 071 transconjugants; lane 6,

negative control.

4.3.7.2. AHL production by transconjugant strains

Enterobacter sp. 067 transconjugant was not able to induce E. coli pSB403 as

the wild type did (Figure 17A and B, Table 8). This result indicates efficient

expression of the aiiA gene, which did not allow the accumulation of AHL

molecules. The bioluminescence sensitivity value determined by a photon-counting

camara was of 9.6 for the Enterobacter sp. 067 transconjugant (Figure 17A), 6.7 for

the Enterobacter sp. 067 wild type (Figure 17B), and 3.0 for the positive control, B.

cepacia H111 (Figure 17C). Low sensitivity values of bioluminescence indicate the

presence of AHLs in the medium, while high values indicate that these molecules are

absent.

However, the mechanism of quorum quenching did not influence the AHL

production by H. alvei 068 and 071. These transconjugants were able to grow in LB

supplemented with kanamycin, but they accumulated AHLs. As they have a native

plasmid, the triparental mating approach did not work well with these strains. It is

probable that some recombination between pBHR1-aiiA-Km

and the native plasmid

occurred, resulting in inhibition of aiiA gene expression. Another possibility could be

that these strains produced high amounts of AHLs and the concentration of lactonase

in the cells was insufficient to completely degrade these molecules.

500

1000

1 2 3 4 5 6 7

10000

250

750

1500

2000

129

Figure 17 – Activation of the AHL monitor strain E. coli pSB403 in cross-streak

experiment. (A) E. coli pSB403 cross-streaked to 067 transconjugant;

(B) E. coli pSB403 cross-streaked to 067 wild type; (C) positive

control, E. coli pSB403 cross-streaked to B. cepacia H111.

Table 8 – Values of bioluminescence produced by E. coli MT102 pSB403 at 100 nm

after growth in LB broth supplemented with the supernatant of

Enterobacter sp. 067 wild type, Enterobacter sp. 067 transconjugant, or

with HHL standard. Data represent average of triplicate experiments.

Bioluminescence values

Dilution rate

067

Wild type

067-aiiA

Transconjugant

HHL

Positive control

Negative control

1/2 30041

134

26989 174

1/4 6288

123

22226 145

1/8 184

127

995 129

1/16 123

104

864 123

1/32 123

105

264 105

1/64 119

109

252 103

1/128 113

172 119

1/256 105

139 108

4.3.7.3. Proteolytic activity of wild type and transconjugants

Enterobacter sp. 067 transconjugant was more proteolytic than the wild type

in reconstituted milk (Figure 18) and on LB agar supplemented with 2% skim milk

powder (Figure 19). Higher protease activity by a transconjugant strain may occur

due to the inability of the HalR protein to interfere with protease expression when it

B A

130

is bound to an AHL. Another possibility is related to the production of more than one

protease that is not negatively regulated by quorum sensing. Production of protease

by Enterobacter sp. 067 wild type indicated that AHL did not completely repress

HalR binding to the protease promoter. This result suggests a negative regulation of

the quorum sensing mechanism in this strain, which needs further investigation.

Figure 18 – Proteolytic activity in 12% (w/v) reconstituted skim milk powder

inoculated with Enterobacter sp. after incubation for 24 h at 30 °C.

Enterobacter sp. transconjugant, (067) Enterobacter sp. wild type.

Figure 19 – Proteolytic activity on LB agar supplemented with 2% (w/v) skim milk

powder after incubation for 24 h at 30 °C. Clearing zones are indicative

of protease activity. (067) Enterobacter sp. wild type, (067-7)

Enterobacter sp. transconjugant, (067-10) Enterobacter sp.

transconjugant.

While most LuxR-type proteins act as AHL-dependent transcriptional

activators, a few members appear to act as repressors. Their ability to repress

067 aiiA 067

067

067-7

067

067-10

131

transcription is not surprising given that LuxR and TraR can act as repressors when

their binding sites are moved. What is striking, however, is that AHLs abolish

repression and, in at least one case, abolish DNA binding (VON BODMAN et al.,

1998; NASSER et al., 1998). In this regard, these repressor proteins work in

fundamentally different ways from the majority of LuxR-type proteins. The best

characterized examples of repression by AHLs are the EsaR/EsaI system of Pantoea

stewartii (VON BODMAN et al., 1998), a vascular pathogen of maize, and the

ExpR/ExpI system of Erwinia chrysanthemi, another plant pathogen that macerate

plant tissues by releasing pectinases and other hydrolytic enzymes. Mutations in esaI

abolish exopolysaccharide production and prevent pathogenesis (VON BODMAN et

al., 1998). In contrast, mutations in esaR cause hyperproduction of

exopolysaccharides. The double mutant has the esaR phenotype. These data suggest

that EsaR is a direct repressor of genes required for exopolysaccharide production.

However, this protein could also act indirectly, for example, as an activator of an

uncharacterized repressor gene. On the other hand, it seems inescapable that the

AHL made by EsaI (3-oxo-hexanoyl-HSL) must oppose EsaR function rather than

stimulate it. Evidence that EsaR is a direct repressor was obtained from studies of the

esaR promoter, which is autorepressed by EsaR. Purified EsaR bound to the esaR

promoter in the absence of AHL, but not in its presence. Like EsaR, ExpR of E.

chrysanthemi also autorepresses its synthesis in the absence of AHL (also 3-oxo-

hexanoyl-HSL) and this DNA binding is inhibited by AHL (NASSER et al., 1998).

Gel shift assays and footprinting assays showed that ExpR acts directly upon its

promoter. Both EsaR and ExpR could in principle act as activators of other

promoters, but seems likely that AHL would oppose protein function (by abolishing

DNA binding) at all target promoters. It is fascinating that AHLs could block this

group of proteins from binding DNA, while stimulating DNA binding of other LuxR-

type proteins.

In a preliminary assay, H. alvei 068 and 071 wild type showed the same

proteolytic activity as the H. alvei 068 and 071 transconjugants. As the quorum

quenching mechanism did not compromise accumulation of AHLs by H. alvei 068

and 071, it was not possible to evaluate the phenotypes controlled by quorum sensing

in these strains. Bruhn et al. (2004) made comparisons between H. alvei wild type,

the AHL-negative mutant, and the OHHL-complemented AHL-negative mutant in

many assays and no difference was found between the strains in the following tests:

132

production of antibiotics, biogenic amines, adhesion and biofilm formation, motility,

starvation survival, resistance to oxidative stress, or virulence against Drosophila.

This suggests that these phenotypes are not upon quorum sensing regulation and

more studies need to be done to understand the role of AHLs in this species.

On the other hand, Viana (2006) verified the inhibitory effect of furanones, a

quorum sensing inhibitor, on biofilm formation by H. alvei 071. Then, the

development of mutants of H. alvei is important in order to confirm the phenotypes

regulated by quorum sensing in this bacterium.

4.3.7.4. Influence of quorum quenching mechanism in expression of

extracellular proteins by

Enterobacter

sp. 067

As the transconjugant strain was more proteolytic than the wild type, SDS-

PAGE and zymogram were developed to evaluate if there were differences in the

pattern of extracellular proteins between them. Only one protein was missing in the

transconjugant strain (Figure 20A) and this might have occurred due to the control of

quorum sensing in expression of this protein.

Although three different proteins were detected in the concentrated

supernatant of 067 wild type inoculated in TYEP medium (Figure 20A), none of

them showed proteolytic activity in the SDS-PAGE supplemented with azocasein

(Figure 20B). This confirmed the previous result of absence of proteolytic activity in

the supernatant of TYEP medium inoculated with Enterobacter sp. 067. This might

occur because this strain does not produce and secrete a protease into TYEP,

azocasein is not the best substrate for protease, or the process of renaturation of this

enzyme after development of SDS-PAGE was not sufficient.

The different proteins of wild type 067 present in the SDS-PAGE (Figure

20A) were purified from the gel and analysed by mass spectrometry. Only the

protein of approximately 50 kDa was identified and showed identity to FliC, a

bacterial flagellin, from E. coli (GenBank accession number Q842A7).

133

Figure 20 – SDS-PAGE (A) and zymogram azocasein (B) gels (12%) showing

extracellular protein production by Enterobacter sp. 067 after growth

for 18 h at 30 °C in TYEP medium supplemented with 0.25% CaCl

(S) molar mass standards (BioRad); (WT) wild type; (T)

transconjugant; (SU) supernatant of TYEP medium; (C) supernatant

of TYEP medium concentrated four times; (F) filtrate of TYEP

medium after concentration.

4.3.8. Native plasmid of H. alvei

Once a native plasmid was detected in H. alvei 068 and 071 after restriction

of pBHR1-aiiA-Km

extracted from the transconjugant strains, wild type strains were

grown and the plasmid was extracted and loaded on an agarose gel. It was confirmed

that H. alvei 068 and 071 have a native plasmid that was named pMLM (Figure 21).

The size of this plasmid extracted from wild type H. alvei 068 and 071 was

different from the size observed when it was extracted from the transconjugant

strains. This might indicate that some rearrangement occurred in this molecule after

the triparental mating.

250

100

S WT T WT T WT T

150

S WT T WT T WT T

250

100

134

Figure 21 – Agarose gel electrophoresis of pMLM extraction. Lane 1, pMLM068;

lane 2, pMLM071.

A total of 32 restriction enzymes were used to digest pMLM and only DdeI,

HinfI, MspI, and RsaI were able to cut this plasmid. In order to clone and sequence

the DNA fragments, RsaI was used to digest pMLM since it produces blunt ends

after digestion and because, afterwards, the Zero Blunt

TOPO

PCR Cloning Kit

(Invitrogen, United Kingdom) was used.

A fragment of 1,857 bp of pMLM was sequenced and sites for three

endonucleases, ClaI, ScaI, and KpnI, which were used to digest this plasmid

previously, were found (Figure 22). However, these restricition enzymes did not

cleave pMLM, probably due to methylation in the sites of these endonucleases.

The plasmid pMLM appears to be a new molecule and its complete sequence

needs to be elucidated. It did not show similarity with the plasmids pAlvA and

pAlvB, which contain genes that encode alveicin, a bacteriocin from H. alvei (Wertz

and Riley, 2004). Plasmids pAlvA and pAlvB resemble the ColE1-type replicons and

carry mobilization genes, as well as colicin-like bacteriocin operons. Plasmid pAlvA

is 5113 bp, 46.18% G+C, and was sequenced from five of six H. alvei strains

examined by Wertz and Riley (2004). Plasmid pAlvB is 5216 bp, 46.97% G+C, and

was isolated from strain MISC261 (WERTZ and RILEY, 2004).

135

Figure 22 – Restriction map of a fragment of 1,857 bp fragment of pMLM

constructed using Clone Manager 5.0. *Enzymes previously used to

digest pMLM that did not cleave it.

4.3.9. Amplification of AHL synthase (halI) and AHL receptor (halR) genes by

PCR

Primers based on the sequences of the halI and halR genes of H. alvei were

constructed and used to amplify these genes by PCR. Amplified products of the

expected sizes, 660 bp or 751 bp, were obtained for the halI (Figure 23) and halR

(Figure 24) genes, respectively.

The halI gene was detected in all strains of H. alvei used in this study, as well

as in Enterobacter sp. 067 (Figure 23). The halI gene of H. alvei 068 and 071

showed 99% identity with each other. The same result was observed when the halI

and halR genes of H. alvei 068 were compared to the halI and halR genes of

Enterobacter sp. 067. However, when sequences of the halI gene of H. alvei 068 and

H. alvei 059 were aligned, they showed only 75% identity with each other.

136

Figure 23 – Agarose gel electrophoresis of PCR products obtained from halI gene

amplification. (S) standard, (059) H. alvei, (067) Enterobacter sp.,

(068) H. alvei, (071) H. alvei, (B) negative control: reaction mix

without DNA.

Figure 24 – Agarose gel electrophoresis of PCR products obtained from halR gene

amplification. (S) standard, (067) Enterobacter sp., (068) H. alvei, (B)

negative control negative control: reaction mix without DNA.

Amino acid sequence alignments of LuxI-type proteins revealed ten

completely conserved amino acids, most of these within the amino-terminal halves of

the proteins. Of the ten conserved amino acid residues, seven are charged. The

observation that the amino-terminal portions of LuxI-type proteins are the best

conserved, whereas the carboxy-termini are more divergent, suggests that the C-

termini may provide recognition of the different acyl chains on precursor acyl-ACPs

(FUQUA et al., 2001).

750

1000

S 059 067 068 071 B S

250

500

1500

750

1500

S 067 068 B

250

500

1000

2000

137

On the other hand, according to Fuqua et al. (2001), LuxR-type proteins share

an end-to-end sequence identity of 18-23%. An acyl-HSL interaction region

(residues 66 to 138) and a DNA binding motif (residues 183-229) are defined by two

clusters of stronger sequence conservation. LuxR-type proteins are members of the

larger FixJ-NarL superfamily. Most members of the FixJ-NarL superfamily are two

component-type response regulators that differentially control DNA binding activity

by phosphorylation of a conserved aspartate residue in the amino-terminal halves of

these proteins. There is no significant sequence similarity between the amino-

terminal halves of LuxR-type proteins and other members of the FixJ-NarL group.

This reflects the specific function of this region in the acyl-HSL interaction of LuxR-

type proteins. However, several mechanistic features may be shared between LuxR-

type proteins and other members of the FixJ-NarL superfamily. LuxR-type proteins

facilitate responses to acyl-HSLs through a series of recognizable steps, including (1)

specific binding of cognate acyl-HSLs, (2) conformational changes and alterations in

multimerization of the protein following binding of the signal, (3) binding or release

of specific regulatory sequences upstream of the target genes, and often, (4)

activation of transcription.

4.3.10. Sequencing and overexpression of halI in E. coli XL1-Blue

In order to know which AHL molecules are synthesized by HalI, the halI

gene was sequenced, cloned (Figure 25) and overexpressed in E. coli XL1-Blue. This

gene comprised 660 bp and encoded a protein with 216 amino acids. The protein

length of HalI was in agreement with LuxI-type proteins, which range from 194 to

226 amino acids long (FUQUA et al., 2001). This protein has a predicted molecular

mass of 25 kDa but based on electrophoretic mobility, the molecular mass was

estimated to be approximately 16 kDa (Figure 26). This result suggests possible

degradation of HalI.

138

Figure 25 – Agarose gel electrophoresis of restriction reactions of pQE-30Xa-halI

containing the cloned halI gene. (S) Standard; (1, 2, 3, 4, 5, 6) clones of

E. coli XL1-Blue pQE-30Xa-halI.

Figure 26 - SDS-PAGE (12%) showing AHL synthase production after expression of

6xHis-tagged HalI from H. alvei 068 in E. coli XL1-Blue. (S) molar mass

standards (BioRad); (1 and 3) crude extracts of E. coli XL1-Blue pQE-

30Xa-halI; (2 and 4) crude extracts of E. coli XL1-Blue containing the

plasmid pQE-30Xa.

S 1 2 3 4

100

150

250

500

1000

1500

S 1 2 3 4 5 6

pQE-30XA

halI

250

750

2000

10000

3500

139

4.3.11. Detection of AHL molecules produced by E. coli XL1-Blue pQE-30Xa-

halI

4.3.11.1. Cross-streak assay

A bioluminescence sensitivity of 3.9 detected when E. coli pSB403 was

cross-streaked to E. coli XL1-Blue pQE-30Xa-halI indicates successfull heterologous

expression of HalI in E. coli (Figure 27A). The positive control, B. cepacia H111,

resulted in a bioluminescence sensitivity, measured by photon-counting camera, after

induction of E. coli pSB403 equal to 3.8 (Figure 27B) and the negative control, E.

coli XL1-Blue pQE-30Xa, resulted in a bioluminescence induction of 10, indicating

that it did not produce AHLs (Figure 27C).

Figure 27 – Activation of the AHL monitor strain E. coli pSB403 in cross-streak

experiments. (A) E. coli pSB403 cross-streaked to E. coli XL1-Blue

pQE-30Xa-halI; (B) E. coli pSB403 cross-streaked to B. cepacia H111,

positive control; (C) E. coli pSB403 cross-streaked to E. coli XL1-Blue

pQE-30Xa, negative control.

4.3.11.2. Thin layer chromatography assay

Supernatant extracts of E. coli XL1-Blue harboring pQE-30Xa-halI cultured

in ABG media induced E. coli pSB403 and C. violaceum CV026 on the TLC assays

(Figures 28 and 29). The spots observed on TLC, on which E. coli pSB403 was used

as a biosensor, presented the same retention factor (rf) as 3-oxo-C6-HSL (Figure 28),

while a small spot close to C6-HSL was detected on TLC developed with C.

140

violaceum CV026 (Figure 29). These results indicated that the halI gene encodes the

AHL synthase able to synthesize 3-oxo-C6-HSL and C6-HSL.

These results were confirmed with the supernatant extracts of dYT medium.

Figure 28 - A representative thin-layer chromatogram of HalI expression in E. coli

XL1-Blue cultured in ABG minimal medium. The spots were detected

with E. coli pSB403 reporter strain. Standards: N-(3-oxohexanoyl)-L-

homoserine lactone (OHHL), N-(hexanoyl)-L-homoserine lactone

(HHL); N-(octanoyl)-L-homoserine lactone (OHL); (068) H. alvei wild

type, AHL extract diluted 50 times in ethyl acetate; (HalI) E. coli XL1-

Blue harbouring pQE-30Xa-halI.

Figure 29 - A representative thin-layer chromatogram of HalI expression in E. coli

XL1-Blue cultured in ABG minimal medium. Spots were detected with

C. violaceum CV026 reporter strain. Standards: N-(3-oxohexanoyl)-L-

homoserine lactone (OHHL); N-(hexanoyl)-L-homoserine lactone

(HHL); N-(octanoyl)-L-homoserine lactone (OHL); (HalI) E. coli XL1-

Blue harbouring pQE-30Xa-halI.

OHHL HHL/OHL 068 HalI

1 µl 10 µl

OHHL HHL/OHL HalI

20 µl

141

4.3.11.3. Chemical characterization of AHL molecules by LC-MS

In heterologous expression of HalI protein, the extract of AHL molecules

obtained from ABG minimal medium inoculated with E. coli XL1-Blue pQE-30Xa-

halI was analysed by LC-MS and C6-HSL and 3-oxo-C6-HSL were detected (Figure

30), confirming the preliminary results of the TLCs. Interestingly, another

noteworthy result is the observation that the H. alvei parental strain produces 3-oxo-

C6-HSL, C6-HSL, 3-oxo-C8-HSL, and C8-HSL and the halI gene, when expressed

in E.coli, directed the synthesis only of two of these molecules. This suggests that

there is a gene encoding a second putative AHL synthase which is significantly

different from halI or E. coli was not able to synthesize 3-oxo-C8-HSL and C8-HSL.

A known example of bacteria that has two AHL synthases is P. aeruginosa.

The LasI/LasR system of this bacterium, which is homologue of LuxI/LuxR, initiates

a cascade of signaling by the induction of transcription of virulence factors in high

cell density. LasI synthesizes an AHL known as 3-oxo-C12-HSL (PASSADOR et

al., 1993). Moreover, LasI/LasR activates expression of rhlI and rhlR, which activate

genes that are already under the control of LasI/LasR, besides some additional genes.

The protein RhlI synthesizes a second AHL molecule known as C4-HSL (LATIFI et

al., 1995). The regulation of RhlI/RhlR by LasI/LasR ensures the establishment of

two quorum sensing circuits that occur in a sequential way in P. aeruginosa.

Figure 30 - High-performance liquid chromatography-positive electrospray

ionization (ESI

)-MS chromatogram showing the mass spectra for

the signal molecules present in cell free supernatant of E. coli XL1-

Blue pQE-30Xa-halI. Signal molecule extract was obtained from

overnight cell free culture supernatant in ABG minimal medium.

Time

4681012

Standard

pQE Hal I

3-oxo C6

Time

46810 12

Standard

pQE Hal I

3-oxo C6

142

4.3.12. Detection of auto-inducer two (AI-2)

Sterilized supernatant of overnight culture of A. hydrophila 099 in LB broth

was able to induce bioluminescence production by V. harveyi BB170 (Table 9). This

result indicates production of AI-2, although in the literature there isn’t any data

related to production of this molecule by A. hydrophila.

While the AHLs are confined to a reasonable range of bacteria, recent

evidence has suggested the existence of a universal quorum sensing language. A

family of molecules, termed AI-2, is common to many gram-negative and gram-

positive pathogens. However, there is no direct evidence for the involvement of AI-2

in the regulation of pathogenic traits. Furthermore, whether AI-2 has a true role in

quorum sensing signaling in general has been questioned, with suggestions that in

most bacteria AI-2 is simply a metabolic byproduct, which casts doubt on its

suitability as a target in the context of quorum sensing inhibition (CÁMARA et al.,

2002).

Table 9 – Detection of auto-inducer two in supernatant of LB medium inoculated

with psychrotrophic strains. V. harveyi BB170 was used as a monitor

strain and V. harveyi BB120 was used as a positive control.

Strains and medium Luminescence at 175 nm*

Pantoea sp. 039 1973 ± 345

H. alvei 059 2948 ± 810

Enterobacter sp 067 2087 ± 439

H. alvei 068 2899 ± 606

H. alvei 071 3708 ± 687

A. hydrophila 099 12903 ± 192

V. harveyi BB120 4478 ± 390

LB medium 2299 ± 384

AB medium 1927 ± 336

Average and standard deviation of data is shown. n: number of repetitions equal 8.

4.3.13. Pathogenesis against Caenorhabditis elegans

As the bacteria used in this study are recognized as opportunistic pathogens,

assays were developed using the nematode C. elegans, which is a valuable model for

studying pathogenesis of various species of bacteria (EWBANK, 2002; CARDONA

143

et al., 2005). It was observed that only A. hydrophila was able to kill 100% of

C. elegans (Figure 31). H. alvei 059, 068, and 071 were not pathogenic to this worm

and Enterobacter sp. 067 and Pantoea sp. 039 were able to kill 25% and 28% of the

nematodes, respectively, after 48 h of incubation (Figure 31).

The pathogenicity scores were based on the percent survival and appearance

of worms at two days post-infection and the total number of parental and progeny

nematodes after five days, as described in the material and methods. As controls,

infections with the non-pathogenic E. coli strain OP50 and B. cepacia H111, whose

pathogenic phenotypes were assigned scores of 0 and 3, respectively, were

performed. Among the six psychrotrophic strains screened, only A. hydrophila 099

presented pathogenicity and the score of 3 was comparable to that of B. cepacia

H111 (Table 10).

100

120

B. cepacia H1

E. coli O

lve

i 0

Entero

er sp 067

alve

lve

i 0

ila

cter 067 aiiA

Strains

% dead worms

Figure 31 – Pathogenicity of psychrotrophic strains isolated from cooled raw milk in

the C. elegans Bristol N2 model. The strains were grown on NGM agar

plates at 30 °C and the mortality assay was performed.

Proteolytic activity of some extracellular enzymes of A. hydrophila was

considered to play a major role in the virulence and pathogenicity of the bacterium.

These enzymes provide nutrients by breaking down host proteins into small

144

molecules capable of entering the bacterial cell. A. hydrophila is known as a

pathogenic bacterium to C. elegans (EWBANK, 2002). In addition, according to

Kirov (2003), this bacterium is associated with gastroenteritis and its pathogenicity

and virulence to humans depend on the ability to produce factors, such as exotoxins,

cytotoxins, endotoxins, siderophores, invasins, adhesins, S-layers and flagella.

Table 10 – Scores obtained by plate mortality assay

Strain Pathogenicity score

B. cepacia H111 3

E. coli OP50 0

H. alvei 059 0

Enterobacter sp 067 0

H. alvei 068 0

H. alvei 071 0

A. hydrophila 099 3

Enterobacter sp 067-7 0

Pantoea sp 039 0

145

4.4. CONCLUSIONS

The strains 039, 059, 067, 068, 071, and 099 were identified as Pantoea sp.,

H. alvei, Enterobacter sp., H. alvei, H. alvei, and A. hydrophila, respectively, based

on 16S rDNA sequencing. These strains showed different potentials to spoil milk and

only A. hydrophila 099 presented proteolytic and lipolytic activities in supernatants

of TYEP and LB.

H. alvei possesses a native plasmid, the halI and halR genes, and produces 3-

oxo-C6-HSL, C6-HSL, C8-HSL, and 3-oxo-C8-HSL. Besides, the aiiA

transconjugants of H. alvei 068 and 071 still accumulate AHL molecules and the

overexpression of HalI in E. coli suggests the presence of an additional AHL-based

quorum sensing system in H. alvei.

Enterobacter sp. 067 contains the halI and halR genes, produces 3-oxo-C6-

HSL, and C6-HSL, and the transconjugant is more proteolytic than wild type

suggesting that AHL-mediated quorum sensing negatively regulates protease

expression.

A. hydrophila 099 produces C4-HSL, C6-HSL, AI-2 and proved to be

pathogenic in the C. elegans model system.

146

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152

APPENDICES

CHAPTER 2

Table 2.1 - Identification of proteins in ammonium sulfate precipitated supernatant of

P. fluorescens 041 by MALD-TOF mass spectrometry.

Band

Accession Mass

Identified protein

Score Description

Q7X4S5 49440 Extracellular alkaline metalloprotease 64 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 254 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 239 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 148 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 89 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 123 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 56 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 221 P. fluorescens

Q7X4S5 49440 Extracellular alkaline metalloprotease 91 P. fluorescens

1 – Bands were extracted from SDS-PAGE, line 3, figure 2.

153

AprX07A MSKVKDKAIV SAAQASTAYS QIDSFSHLYD RGGNLTVNGK PSYTVDQAAT QLLRDGAAYR 60

AprX041 MSKVKDKAIV SAAQASTAYS QIDSFSHLYD RGGNLTVNGK PSYTVDQAAT QLLRDGAAYR

AY298902 MSKVKDKAIV SAAQASTAYS QIDSFSHLYD RGGNLTVNGK PSYTVDQAAT QLLRDGAAYR

DQ146945 MSKVKDKAIV SAAQASTAYS QIDSFSHLYD RGGNLTVNGK PSYTVDQAAT QLLRDGAAYR

.......... .......... .......... .......... .......... ..........

AprX07A DFDGNGKIDL TYTFLTSATQ STMNKHGISG FSQFNTQQKA QAALAMQSWA DVANVTFTEK 120

AprX041 DFDGNGKIDL TYTFLTSATQ STMNKHGISG FSQFNTQQKA QAALAMQSWA DVANVTFTEK

AY298902 DFDGNGKIDL TYTFLTSATQ STMNKHGISG FSQFNTQQKA QAALAMQSWA DVANVTFTEK

DQ146945 DFDGNGKIDL TYTFLTSATQ STMNKHGISG FSQFNTQQKA QAALAMQSWA DVANVTFTEK

.......... .......... .......... .......... .......... ..........

AprX07A

ASGGDGHMTF GNYSSGQDGA AAFAYLPGTG AGYDGTSWYL TNNSYTPNKT PDLNNYGRQT 180

AprX041

ASGGDGHMTF GNYSGGQDGA AAFAYLPGTG AGYDGTSWYL TNNSYTPNKT PDLNNYGRQT

AY298902

ASGGDGHMTF GNYSSGQDGA AAFAYLPGTG AGYDGTSWYL TNNSYTPNKT PDLNNYGRQT

DQ146945

ASGGDGHMTF GNYSSGQDGA AAFAYLPGTG AGYDGTSWYL TNNSYTPNKT PDLNNYGRQT

.......... ....*..... .......... .......... .......... ..........

AprX07A

LTHEIGHTLG LAHPGDYNAG NGNPTYNDAT YGQDTRGYSP MSYWSESNTN QNLSKGGVEA 240

AprX041

LTHEIGHTLG LAHPGDYNAG NGNPTYNDAT YGQDTRGYSL MSYWSESNTN QNFSKGGVEA

AY298902

LTHEIGHTLG LAHPGDYNAG NGNPTYNDAT YGQDTRGYSL MSYWSESNTN QNFSKGGVEA

DQ146945

LTHEIGHTLG LAHPGDYNAG NGNPTYNDAT YGQDTRGYSL MSYWSESNTN QNFSKGGVEA

.......... .......... .......... .......... .......... ..*.......

AprX07A

YASGPLIDDI AAIQKLYGAN FNTRATDTTY GFNSNTGRDF LSATSNADKL VFSVWDGGGN 300

AprX041

YASGPLIDDI AAIQKLYGAN LSTRATDTTY GFNSNTGRDF LSATSNADKL VFSVWDGGGN

AY298902

YASGPLIDDI AAIQKLYGAN LSTRATDTTY GFNSNTGRDF LSASSNADKL VFSVWDGGGN

DQ146945

YASGPLIDDI AAIQKLYGAN LSTRATDTTY GFNSNTGRDF LSATSNADKL VFSVWDGGGN

.......... .......... **........ .......... ...*...... ..........

AprX07A

DTLDFSGFTQ NQKINLTATS FSDVGGLVGN VSIAKGVTIE NAFGGSGNDL IIGNQVANTI 360

AprX041

DTLDFSGFTQ NQKINLTATS FSDVGGLVGN VSIAKGVTIE NAFGGAGNDL IIGNQVANTI

AY298902

DTLDFSGFTQ NQKINLTATS FSDVGGLVGN VSIAKGVTIE NAFGGSGNDL IIGNQVANTI

DQ146945

DTLDFSGFTQ NQKINLTATS FSDVGGLVGN VSIAKGVTIE NAFGGSGNDL IIGNQVANTI

.......... .......... .......... .......... .....*.... ..........

AprX07A

KGGAGNDLIY GGGGADQLWG GAGSDTFVYG ASSDSKPGAA DKIFDFTSGS DKIDLSGITK 420

AprX041

KGGAGNDLIY GGGGADQLWG GAGSDTFVYG ASSDSKPGAA DKIFDFTSGS DKIDLSGITK

AY298902

KGGAGNDLIY GGGGADQLWG GTGSDTFVYG ASSDSRPGAA DKIFDFTSGS DKIDLSGITK

DQ146945

KGGAGNDLIY GGGGADQLWG GTGSDTFVYG ASSDSRPGAA DKIFDFTSGS DKIDLSGITK

.......... .......... .*........ .....*.... .......... ..........

AprX07A

GAGVTFVNAF TGHAGDAVLT YASGTNLGTL AVDFSGHGVA DFLVTTVGQA AASDIVA 477

AprX041

GAGVTFVNAF TGHAGDAVLS YASGTNLGTL AVDFSGHGVA DFLVTTVGQA AASDIVA

AY298902

GAGVTFVNAF TGHAGDAVLT YASGTNLGTL AVDFSGHGVA DFLVTTVGQA AASDIVA

DQ146945

GAGVTFVNAF TGHAGDAVLT YASGTNLGTL AVDFSGHGVA DFLVTTVGQA AASDIVA

.......... .........* .......... .......... .......... .......

Figure 2.1 – Multiple sequence alignment of deduced protease AprX from

P. fluorescens 07A and 041 (this study), P. fluorescens A506

(Genbank accession number AY298902), and P. fluorescens strain F

(Genbank accession number DQ146945). The differences of

similarity in amino acid residues are indicated by gray shading and

the catalytic domain of neutral zinc metalloprotease is underlined.

154

LipM07A MGVFDYKNLG TEGSKALFAD AMAITLYSYH NLDNGFAVGY QNNGLGLGLP ATLVSALIGG 60

LipM041 MGMFDYKNLG TEDSKALFAD AMAITLYSYH NLDNGFAVGY QNNGLGLGLP ATLVSALIGG

DQ305493

MGIFDYKNLG TEGSKALFAD AMAITLYSYH NLDNGFAVGY QHNGLGLGLP ATLVGALLGS

AY694785

MGIFDYKNLG TEGSKTLFAD AIAITLYSYH NLDNGFAVGY QHNGLGLGLP ATLVGALLGS

AF216702

MGIFDYKNLG TEGSKTLFAD AMAITLYSYH NLDNGFAVGY QHNGLGLGLP ATLVGALLGS

..*....... ..*..*.... .*........ .......... .*........ ....*..*.*

LipM07A

SNAQGVIPGI PWNPDSEKAA LEAVQAAGWT PISASTLGYS GKVDARGTYF GEKFGYGTAQ 120

LipM041

SNAQGVIPGI PWNPDSEKAA LEAVQAAGWT PISASTLGYS GKVDARGTFF GEKFGYGTAQ

DQ305493

TNSQGVIPGI PWNPDSEKAA LEAVQNAGWT PISASTLGYG GKVDARGTYF GEKAGYTTAQ

AY694785

TDSQGVIPGI PWNPDSEKAA LEAVQKAGWT PISASDLGYG GKVDGRGTFF GEKAGYTTAQ

AF216702

TDSQGVIPGI PWNPDSEKAA LEAVQKAGWT PISASALGYA GKVDARGTFF GEKAGYTTAQ

***....... .......... .....*.... .....*...* ....*...*. ...*..*...

LipM07A

AEVLGKYDDA GKLLEIGISF RGTSGPRESV ITDTIGDVIN DLLAAFGPKD YAKNYAGEAF 180

LipM041

AEVLGKYDDA GKLLEIGISF RGTSGPRESV ITDSIGDVIS DLLAAFGPKD YAKNYAGEAF

DQ305493

VEVLGKYDDA GKLLEIGIGF RGTSGPRETL ISDSIGDLVS DLLAALGPKD YAKNYAGEAF

AY694785

VEVLGKYDDA GKLLEIGIGF RGTSGPRESL ITDSIGDVIS DLLAAFGPKD YAKNYAGEAF

AF216702

VEVLGKYDDA GKLLEIGIGF RGTSGPRETL ISDSIGDLIS DLLAALGPKD YAKNYAGEAF

*......... ........*. ........** .*.*...*** .....*.... ..........

LipM07A

GGLLKNVADY ATAQGLGGND VVVSGHSLGG MAVNSMADLS DSTWSGFYKD ANYVAYASPT 240

LipM041

GGLLKNVADY ATAQGLGGND VVVSGHSLGG LAVNSMADLS DSTWSGFYKD SNYVAYASPT

DQ305493

GGLLKNVADY AAAHGLTGKD VVVSGHSLGG LAVNSMADLS TNKWSGFYTD ANYVAYASPT

AY694785

GGLLKNVADY AGAHGLSGKD VVVSGHSLGG LAVNSMADLS NNKWSGFYKD ANYVAYASPT

AF216702

GGLLKNVADY AGAHGLTGKD VVVSGHSLGG LAVNSMADLS NYKWAGFYKD ANYVAYASPT

.......... .*.*..*.*. .......... *......... ***.*...*. *.........

LipM07A

QSAGDKVLNV GYENDPVFRA LDGSSFNLSS LGVHDKAHES STDNIVSFND HYASSLWNVL 300

LipM041

QSAGDKVLNV GYENDPVFRA LDGSSFNLSS LGVHDKPHES STDNIVSFND HYASSLWNVL

DQ305493

QSAGDKVLNI GYENDPVFRA LDGSSFNLSS LGVHDKPHES TTDNIVSFND HYASTLWNVL

AY694785

QSAGDKVLNI GYENDPVFRA LDGSSFNLSS LGVHDKPHES TTDNIVSFND HYASTLWNIL

AF216702

QSAGDKVLNI GYENDPVFRA LDGSSFNLSS LGVHDKPHES TTDNIVSFND HYASTLWNVL

.........* .......... .......... ......*... *......... ....*...*.

LipM07A

PFSILNLPTW VSHLPTGYGD GMTRILDSGF YEQMTRDSTV IVANLSDPAR ATTWVQDLNR 360

LipM041

PFSILNLPTW VSHLPTGYGD GMTRILDSGF YEQMTRDSTV IVANLSDPAR ATTWVQDLNR

DQ305493

PFSIVNLPTW VSHLPTAYGD GMTRILDSGF YDQMTRDSTV IVANLSDPAR ATTWVQDLNR

AY694785

PFSIVNLPTW VSHLPTGYGD GMTRILESGF YDQMTRDSTV IVANLSDPAR ATTWVQDLNR

AF216702

PFSIVNLPTW VSHLPTAYGD GMTRILESGF YDQMTRDSTV IVANLSDPAR ANTWVQDLNR

....*..... ......*... ......*... .*........ .......... .*........

LipM07A

NAEAHKGNTF IIGSDGDDLI KGGRGADFIE GGKGNDTIRD SSGYNTFLFS GQFGNDRVIG 420

LipM041

NAEAHKGNTF IIGSDGDDLI KGGRGVDFIE GGKGNDTIRD SSGHNTFLFS GQFGNDRVIG

DQ305493

NAEPHKGNTF IIGSDGNDLI QGGKGADFIE GGKGNDTIRD NSGHNTFLFS GQFGNDRVIG

AY694785

NAEPHKSNTF IIGSHGNDLI QGGKGADFIE GGKGNDTIRD NSGHNTFLFS GNFGNDRVIG

AF216702

NAEPHKGNTF IIGSDGNDLI QGGNGADFIE GGKGNDTIRD NSGHNTFLFS GHFGNDRVIG

...*..*... ....*.*... *..*.*.... .......... *..*...... .*........

LipM07A

YQATDKLVFN DVAGSNDYRD HIKVVGGDTV IGFGTDSVTL VGVS--SLSG EGIVIG 474

LipM041

YQATDKLVFN DVAGSTDYRD HAKVVGGDTV ISFGTDSVTL VGVS--SLSG EGIVIG 474

DQ305493

YQTTDKLVFQ DVQGSTDLRD HAKVVGADTV LTFGADSVTL VGVGHGGLWA DGVSIG 476

AY694785

YQTTDKLVFQ NVEGSTDLRD HAKVVGADTV LTFGADSVTL VGVGHGGLWA DGVSIG 476

AF216702

YQPTDKLVFK DVQGSTDLRD HAKVVGADTV LTFGADSVTL VGVGHGGLWT EGVVIG 476

..*......* *.*..*.*.. .*....*... **..*..... ...*--*.** *.**..

Figure 2.2 - Multiple sequence alignment of deduced lipase LipM from P.

fluorescens 07A and 041 (this study), Lip (Genbank accession number

DQ305493), Lip68 (Genbank accession number AY694785), and

LipA (Genbank accession number AF216702) from P. fluorescens.

The differences of similarity in amino acid residues are indicated by

gray shading and the catalytic domain of serine lipase is underlined.

155

CHAPTER 3

Table 3.1 – Composition of LB soft agar

Component Weight (g) and volume (ml)

Tryptone 1.50

Yeast extract 0.75

NaCl 0.75

Agar 1.00

Water 150

Table 3.2 – Composition of AB soft agar for Agrobacterium tumefaciens

Component Volume (ml) and weight (g)

20X AB salt

20X AB buffer

Manitol 10% 5

Water 88

Agar 1

(a) 20X AB salt: NH

Cl (20 g); MgSO

x 7H

O (6 g); KCl (3 g); CaCl

(0.2 g); FeSO

x 7 H

O (0.05

g); water (1000 ml). (b) 20X AB buffer: K

HPO

(60 g); NaH

(23 g); water (1000 ml). The pH

was adjusted to 7.0.

Table 3.3 – Composition of the AB medium for V. harveyi (Bassler et al., 1994)

Component Concentration

NaCl 0.30 M

MgSO

0.05 M

Vitamin-free casamino acids 0.2 %

The pH was adjusted to 7.5 with KOH. The medium was sterilized, cooled, and added of 10 ml of

sterile 1 M potassium phosphate (pH 7.0), 10 ml of 0.1 M L-arginine, 20 ml of glycerol, 1 ml of 10

μg/ml riboflavin, and 1 ml of 1 mg/ml thiamine per litre.

156

CHAPTER 4

Table 4.1 – Composition of the ABC or ABG minimal medium

Component Weight (g) and volume (ml)

A10

(NH

)

HPO

NaCl 30

Destilled water 1000

MgCl

x 6 H

O (1M) 2.0

CaCl x 2 H

O (0.5M) 0.2

FeCl

x 6 H

O (0.01M) 0.3

Destilled water 900

Citrate or glucose 1M

Sterilize the media A10 and B separately at 121 °C per 15 min. Wait cooling to approximately 50 °C.

Sterilize by filtration the citrate or glucose solution. Combine 900 ml of medium B with 100 ml of

medium A10 plus 10 ml of citrate. After preparation, the medium B needs to be maintained in a dark

place.

Table 4.2 – Composition of medium 884 – Tween 80-Agar

Component Weight (g) and volume (ml)

Solution A

Peptone 10.0

NaCl 5.00

CaCl

x 2 H

O 0.10

Agar 15.0

Destilled water 900

Solution B

Tween 80 10.0

Destilled water 100.0

Autoclave solutions A and B separately and combine after cooling to approximately 50 °C. Tween 80

must be weight.

157

Table 4.3 – Composition of NGM I medium

Component Weight (g) and volume (ml)

NaCl 1.50

Tryptone 1.25

Agar 8.50

Destilled water 500

Nystatine (10 mg/ml) 2.50

1 M KPO

buffer, pH 6.0 12.50

1 M CaCl

0.50

1 M MgSO

0.50

Uracile 2 mg/ml (sterile filtrated) 0.50

Cholesterine (10 mg/ml in ethanol) 0.25

Table 4.4 – Composition of NGM II medium

Component Weight (g) and volume (ml)

NaCl 1.50

Bactopeptone 1.75

Agar 8.50

Destilled water 500

Nystatine (10 mg/ml) 2.50

1 M KPO

buffer, pH 6.0 12.50

1 M CaCl

0.50

1 M MgSO

0.50

Uracile 2 mg/ml (sterile filtrated) 0.50

Cholesterine (10 mg/ml in ethanol) 0.25

Table 4.5 – Composition of M9-buffer

Component Weight (g) and volume (ml)

3.0

HPO

6.0

NaCl 5.0

1 M MgSO

1.0

Destilled water 1000

pH 6.0

Table 4.6 – Composition of sodiumhypochlorite solution

Component Volume (µl)

Sterile destilled water 600

Sodiumhypochlorite (12%) 500

NaOH 6N 400

158

039 AGAGTTTGAT TATGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAAC 60

039 GGTAGCACAG AGAGCTTGCT CTCGGGTGAC GAGTGGCGGA CGGGTGAGTA ATGTCTGGGA 120

039 AACTGCCTGA TGGAGGGGGA TAACTACTGG AAACGGTAGC TAATACCGCA TAACGTCGCA 180

039 AGACCAAAGA GGGGGACCTT CGGGCCTCTT GCCATCAGAT GTGCCCAGAT GGGATTAGCT 240

039 AGTAGGTGGG GTAACGGCTC ACCTAGGCGA CGATCCCTAG CTGGTCTGAG AGGATGACCA 300

039 GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG GGGAATATTG 360

039 CACAATGGGC GCAAGCCTGA TGCAGCCATG CCGCGTGTAT GAAGAAGGCC TTCGGGTTGT 420

039 AAAGTACTTT CAGCGGGGAG GAAGGTGTTG TGGTTAATAA CCACAGCAAT TGACGTTACC 480

039 CGCAGAAGAA GCACCGGCTA ACTCCGTGCC AGCAGCCGCG GTAATACGGA GGGTGCAAGC 540

039 GTTAATCGGA ATTACTGGGC GTAAAGCGCA CGCAGGCGGT CTGTCAAGTC GGATGTGAAA 600

039 TCCCCGGGCT CAACCTGGGA ACTGCATTCG AAACTGGCAG GCTAGAGTCT TGTAGAGGGG 660

039 GGTAGAATTC CAGGTGTAGC GGTGAAATGC GTAGAGATCT GGAGGAATAC CGGTGGCGAA 720

039 GGCGGCCCCC TGGACAAAGA CTGACGCTCA GGTGCGAAAG CGTGGGGAGC AAACAGGATT 780

039 AGATACCCTG GTAGTCCACG CCGTAAACGA TGTCGACTTG GAGGTTGTGC CCTTGAGGCG 840

039 TGGCTTCCGG AGCTAACGCG TTAAGTCGAC CGCCTGGGGA GTACGGCCGC AAGGTTAAAA 900

039 CTCAAATGAA TTGACGGGGG CCCGCACAAG CGGTGGAGCA TGTGGTTTAA TTCGATGCAA 960

039 CGCGAAGAAC CTTACCTACT CTTGACATCC AGAGAACTTT CCAGAGATGG ATTGGTGCCT 1020

039 TCGGGAACTC TGAGACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTTGTG AAATGTTGGG 1080

039 TTAAGTCCCG CAACGAGCGC AACCCTTATC CTTTGTTGCC AGCGGTCCGG CCGGGAACTC 1140

039 AAAGGAGACT GCCAGTGATA AACTGGAGGA AGGTGGGGAT GACGTCAAGT CATCATGGCC 1200

039 CTTACGAGTA GGGCTACACA CGTGCTACAA TGGCGCATAC AAAGAGAAGC GACCTCGCGA 1260

039 GAGCAAGCGG ACCTCATAAA GTGCGTCGTA GTCCGGATTG GAGTCTGCAA CTCGACTCCA 1320

039 TGAAGTCGGA ATCGCTAGTA ATCGTAGATC AGAATGCTAC GGTGAATACG TTCCCGGGCC 1380

039 TTGTACACAC CGCCCGTCAC ACCATGGGAG TGGGTTGCAA AAGAAGTAGG TAGCTTAACC 1440

039 TTCGGGAGGG CGCTTACCAC TTTGTGATTC ATGACTGGGG TGAAGTCGTA ACAAGGTAAC 1500

039 CGTAGGGGAA CCTGCGGTTG GATCACCTCC TTT 1533

Figure 4.1 – Nucleotide sequence of 16S rDNA of Pantoea sp. 039.

059 AGAGTTTGAT CCTGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAGC 60

059 GGTAGCACAA GAGAGCTTGC TCTCTGGGTG ACGAGCGGCG GACGGGTGAG TAATGTCTGG 120

059 GAAACTGCCT GATGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATGACGTCT 180

059 TCGGACCAAA GTGGGGGACC TTCGGGCCTC ACGCCATCAG ATGTGCCCAG ATGGGATTAG 240

059 CTAGTAGGTG GGGTAATGGC TCACCTAGGC GACGATCTCT AGCTGGTCTG AGAGGATGAC 300

059 CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT 360

059 TGCACAATGG GCGCAAGCCT GATGCAGCCA TGCCGCGTGT ATGAAGAAGG CCTTCGGGTT 420

059 GTAAAGTACT TTCAGCGAGG AGGAAGGCAT TGTGGTAAAT AACCGCAGTG ATTGACGTTA 480

059 CTCGCAGAAG AAGCACCCGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA 540

059 GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTTGATTAAG TCAGATGTGA 600

059 AATCCCCGAG CTTAACTTGG GAACTGCATT TGAAACTGGT CAGCTAGAGT CTTGTAGAGG 660

059 GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG 720

059 AAGGCGGCCC CCTGGACAAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA 780

059 TTAGATACCC CTGGTAGTCC ACGCTGTAAA CGATGTCGAC TTGGAGGTTG TGCCCTTGAG 840

059 GCGTGGCTTC CGGAGCTAAC GCGTTAAGTC GACCGCCTGG GGAGTACGGC CGCAAGGTTA 900

059 AAACTCAAAT GAATTGACGG GGGCCCGCAC AAGCGGTAGA GCATGTGGTT TAATTCGATG 960

059 CAACGCGAAG AACCTTACCT ACTCTTGACA TCCAGAGAAT TTGCTAGAGA TAGCTTAGTG 1020

059 CCTTCGGGAA CTCTGAGACA GGTGCTGCAT GGCTGTCGTC AGCTCGTGTT GTGAAATGTT 1080

059 GGGTTAAGTC CCGCAACGAG CGCAACCCTT ATCCTTTGTT GCCAGCGCGT AATGGCGGGA 1140

059 ACTCAAAGGA GACTGCCGGT GATAAACCGG AGGAAGGTGG GGATGACGTC AAGTCATCAT 1200

059 GGCCCTTACG AGTAGGGCTA CACACGTGCT ACAATGGCAT ATACAAAGAG AAGCGAACTC 1260

059 GCGAGAGCAA GCGGACCTCA TAAAGTATGT CGTAGTCCGG ATTGGAGTCT GCAACTCGAC 1320

059 TCCATGAAGT CGGAATCGCT AGTAATCGTA GATCAGAATG CTACGGTGAA TACGTTCCCG 1380

059 GGCCTTGTAC ACACCGCCCG TCACACCATG GGAGTGGGTT GCAAAAGAAG TAGGTAGCTT 1440

059 AACCTTCGGG AGGGCGCTTA CCACTTTGTG ATTCATGACT GGGGTGAAGT CGTAACAAGG 1500

059 TAACCGTAGG GGAACCTGCG GTTGGATCAC CTCCTTTCTG 1540

Figure 4.2 – Nucleotide sequence of 16S rDNA of H. alvei. 059

159

067 AGAGTTTGAT CATGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAGC 60

067 GGTAGCACAA GAGAGCTTGC TCTCTGGGTG ACGAGCGGCG GACGGGTGAG TAATGTCTGG 120

067 GAAACTGCCT GACGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATAACGTCG 180

067 CAAGACCAAA GAGGGGGACC TTCGGGCCTC TTGCCATCAG ATGTGCCCAG ATGGGATTAG 240

067 CTAGTAGGTG GGGTAACGGC TCACCTAGGC GACGATCCCT AGCTGGTCTG AGAGGATGAC 300

067 CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT 360

067 TGCACAATGG GCGCAAGCCT GATGCAGCCA TGCCGCGTGT ATGAAGAAGG CCTTCGGGTT 420

067 GTAAAGTACT TTCAGCGGGG AGGAAGGTGT TGTGGTTAAT AACCACAGCA ATTGACGTTA 480

067 CCCGCAGAAG AAGCACCGGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA 540

067 GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTCTGTCAAG TCGGATGTGA 600

067 AATCCCCGGG CTCAACCTGG GAACTGCATT CGAAACTGGC AGGCTAGAGT CTTGTAGAGG 660

067 GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG 720

067 AAGGCGGCCC CCTGGACAAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA 780

067 TTAGATACCC TGGTAGTCCA CGCCGTAAAC GATGTCGACT TGGAGGTTGT GCCCTTGAGG 840

067 CGTGGCTTCC GGAGCTAACG CGTTAAGTCG ACCGCCTGGG GAGTACGGCC GCAAGGTTAA 900

067 AACTCAAATG AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGATGC 960

067 AACGCGAAGA ACCTTACCTA CTCTTGACAT CCAGAGAACT TTCCAGAGAT GGATTGGTGC 1020

067 CTTCGGGAAC TCTGAGACAG GTGCTGCATG GCTGTCGTCA GCTCGTGTTG TGAAATGTTG 1080

067 GGTTAAGTCC CGCAACGAGC GCAACCCTTA TCCTTTGTTG CCAGCGGTCC GGCCGGGAAC 1140

067 TCAAAGGAGA CTGCCAGTGA TAAACTGGAG GAAGGTGGGG ATGACGTCAA GTCATCATGG 1200

067 CCCTTACGAG TAGGGCTACA CACGTGCTAC AATGGCGCAT ACAAAGAGAA GCGACCTCGC 1260

067 GAGAGCAAGC GGACCTCATA AAGTGCGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC 1320

067 CATGAAGTCG GAATCGCTAG TAATCGTAGA TCAGAATGCT ACGGTGAATA CGTTCCCGGG 1380

067 CCTTGTACAC ACCGCCCGTC ACACCATGGG AGTGGGTTGC AAAAGAAGTA GGTAGCTTAA 1440

067 CCTTCGGGAG GGCGCTTACC ACTTTGTGAT TCATGACTGG GGTGAAGTCG TAACAAGGTA 1500

067 ACCGTAGGGG AACCTGCGGT TGGATCACCT CCTTTATG 1538

Figure 4.3 – Nucleotide sequence of 16S rDNA of Enterobacter sp. 067

068 AGAGTTTGAT CATGGCTCAG ATTGAACGCT GGGGGCAGGC CTAACACATG CAAGTCGAGC 60

068 GGTAGCACAA GAGAGCTTGC TCTCTGGGTG ACGAGCGGCG GACGGGTGAG TAATGTCTGG 120

068 GAAACTGCCT GATGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATGACGTCT 180

068 TCGGACCAAA GTGGGGGACC TTCGGGCCTC ACGCCATCAG ATGTGCCCAG ATGGGATTAG 240

068 CTAGTAGGTG GGGTAATGGC TCACCTAGGC GACGATCTCT AGCTGGTCTG AGAGGATGAC 300

068 CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT 360

068 TGCACAATGG GCGCAAGCCT GATGCAGCCA TGCCGCGTGT ATGAAGAAGG CCTTCGGGTT 420

068 GTAAAGTACT TTCAGCGAGG AGGAAGGCAT TAAGGTTAAT AACCTTGGTG ATTGACGTTA 480

068 CTCGCAGAAG AAGCACCGGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA 540

068 GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTTTGTTAAG TCAGATGTGA 600

068 AATCCCCGAG CTTAACTTGG GAACTGCATT TGAAACTGGC AAGCTAGAGT CTTGTAGAGG 660

068 GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG 720

068 AAGGCGGCCC CCTGGACAAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA 780

068 TTAGATACCC TGGTAGTCCA CGCTGTAAAC GATGTCGACT TGAGGTTGTG CCCTTGAGGC 840

068 GTGGCTTCCG GAGCTAACGC GTTAAGTCGA CCGCCTAGGG AGTACGGCCG CAAGGTTAAA 900

068 ACTCAAATGA ATTGACGGGG GCCCGCACAA GCGGTGGAGC ATGTGGTTTA ATTCGATGCA 960

068 ACGCGAAGAA CCTTACCTAC TCTTGACATC CAGAGAATTT GCTAGAGATA GCTTAGTGCC 1020

068 TTCGGGAACT CTGAGACAGG TGCTGCATGG CTGTCGTCAG CTCGTGTTGT GAAATGTTGG 1080

068 GTTAAGTCCC GCAACGAGCG CAACCCTTAT CCTTTGTTGC CAGCACGTGA TGGTGGGAAC 1140

068 TCAAAGGAGA CTGCCGGTGA TAAACCGGAG GAAGGTGGGG ATGACGTCAA GTCATCATGG 1200

068 CCCTTACGAG TAGGGCTACA CACGTGCTAC AATGGCATAT ACAAAGAGAA GCGAACTCGC 1260

068 GAGAGCAAGC GGACCTCATA AAGTATGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC 1320

068 CATGAAGTCG GAATCGCTAG TAATCGTAGA TCAGAATGCT ACGGTGAATA CGTTCCCGGG 1380

068 CCTTGTACAC ACCGCCCGTC ACACCATGGG AGTGGGTTGC AAAAGAAGTA GGTAGCTTAA 1440

068 CCTTCGGGAG GGCGCTTACC ACTTTGTGAT TCATGACTGG GGTGAAGTCG TAACAAGGTA 1500

068 ACCGTAGGGG AACCTGCGGT TGGATCACCT CCTTTCTG 1538

Figure 4.4 – Nucleotide sequence of 16S rDNA of H. alvei 068

160

071 AGAGTTTGAT TATGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAGC 60

071 GGTAGCACAA GAGAGCTTGC TCTCTGGGTG ACGAGCGGCG GACGGGTGAG TAATGTCTGG 120

071 GAAACTGCCT GATGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATGACGTCT 180

071 TCGGACCAAA GTGGGGGACC TTCGGGCCTC ACGCCATCAG ATGTGCCCAG ATGGGATTAG 240

071 CTAGTAGGTG GGGTAATGGC TCACCTAGGC GATGATCTTT AGCTGGTCTG AGAGGATGAC 300

071 CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT 360

071 TGCACAATGG GCGCAAGCCT GATGCAGCCA TGCCGCGTGT ATGAAGAAGG CCTTCGGGTT 420

071 GTAAAGTACT TTCAGCGAGG AGGAAGGCAT TAAGGTTAAT AACCTTGGTG ATTGACGTTA 480

071 CTCGCAGAAG AAGCACCGGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA 540

071 GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTTGATTAAG TCAGATGTGA 600

071 AATCCCCGAG CTTAACTTGG GAACTGCATT TGAAACTGGC AAGCTAGAGT CTTGTAGAGG 660

071 GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG 720

071 AAGGCGGCCC CCTGGACAAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA 780

071 TTAGATACCC TGGTAGTCCA CGCTGTAAAC GATGTCGACT TGGAGGTTGT GCCCTTGAGG 840

071 CGTGGCTTCC GGAGCTAACG CGTTAAGTCG ACCGCCTGGG GAGTACGGCC GCAAGGTTAA 900

071 AACTCAAATG AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGATGC 960

071 AACGCGAAGA ACCTTACCTA CTCTTGACAT CCAGAGAATT TGCTAGAGAT AGCTTAGTGC 1020

071 CTTCGGGAAC TCTGAGACAG GTGCTGCATG GCTGTCGTCA GCTCGTGTTG TGAAATGTTG 1080

071 GGTTAAGTCC CGCAACGAGC GCAACCCTTA TCCTTTGTTG CCAGCACGTG ATGGTGGGAA 1140

071 CTCAAAGGAG ACTGCCGGTG ATAAACCGGG GGAAGGTGGG GATGACGTCA AGTCATCATG 1200

071 GCCCTTACGA GTAGGGCTAC ACACGTGCTA CAATGGCATA TACAAAGAGA AGCGAACTCG 1260

071 CGAGAGCAAG CGGACCTCAT AAAGTATGTC GTAGTCCGGA TTGGAGTCTG CAACTCGACT 1320

071 CCATGAAGTC GGAATCGCTA GTAATCGTAG ATCAGAATGC TACGGTGAAT ACGTTCCCGG 1380

071 GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA 1440

071 ACCTTCGGGA GGGCGCTTAC CACTTTGTGA TTCATGACTG GGGTGAAGTC GTAACAAGGT 1500

071 AACCGTAGGG GAACCTGCGG TTGGATCACC TCCTTTATG 1539

Figure 4.5 – Nucleotide sequence of 16S rDNA of H. alvei 071

099 AGAGTTTGAT TCTGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAGC 60

099 GGCAGCGGGA AAGTAGCTCG CTACTTTTGC CGGCGAGCGG CGGACGGGTG AGTAATGCCT 120

099 GGGAAATTGC CCAGTCGAGG GGGATAACAG TTGGAAACGA CTGCTAATAC CGCATACGCC 180

099 CTACGGGGGA AAGCAGGGGA CCTTCGGGCC TTGCGCGATT GGATATGCCC AGGTGGGATT 240

099 AGCTAGTTGG TGAGGTAATG GCTCACCAAG GCGACGATCC CTAGCTGGTC TGAGAGGATG 300

099 ATCAGCCACA CTGGAACTGA GACACGGTCC AGACTCCTAC GGGAGGCAGC AGTGGGGAAT 360

099 ATTGCACAAT GGGGGAAACC CTGATGCAGC CATGCCGCGT GTGTGAAGAA GGCCTTCGGG 420

099 TTGTAAAGCA CTTTCAGCGA GGAGGAAAGG TTGATGCCTA ATACGTATCA ACTGTGACGT 480

099 TACTCGCAGA AGAAGCACCG GCTAACTCCG TGCCAGCAGC CGCGGTAATA CGGAGGGTGC 540

099 AAGCGTTAAT CGGAATTACT GGGCGTAAAG CGCACGCAGG CGGTTGGATA AGTTAGATGT 600

099 GAAAGCCCCG GGCTCAACCT GGGAATTGCA TTTAAAACTG TCCAGCTAGA GTCTTGTAGA 660

099 GGGGGGTAGA ATTCCAGGTG TAGCGGTGAA ATGCGTAGAG ATCTGGAGGA ATACCGGTGG 720

099 CGAAGGCGGC CCCCTGGACA AAGACTGACG CTCAGGTGCG AAAGCGTGGG GAGCAAACAG 780

099 GATTAGATAC CCTGGTAGTC CACGCCGTAA ACGATGTCGA TTTGGAGGCT GTGTCCTTGA 840

099 GACGTGGCTT CCGGAGCTAA CGCGTTAAAT CGACCGCCTG GGGAGTACGG CCGCAAGGTT 900

099 AAAACTCAAA TGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAT 960

099 GCAACGCGAA GAACCTTACC TGGCCTTGAC ATGTCTGGAA TCCTGTAGAG ATACGGGAGT 1020

099 GCCTTCGGGA ATCAGAACAC AGGTGCTGCA TGGCTGTCGT CAGCTCGTGT CGTGAGATGT 1080

099 TGGGTTAAGT CCCGCAACGA GCGCAACCCC TGTCCTTTGT TGCCAGCACG TAATGGTGGG 1140

099 AACTCAAGGG AGACTGCCGG TGATAAACCG GAGGAAGGTG GGGATGACGT CAAGTCATCA 1200

099 TGGCCCTTAC GGCCAGGGCT ACACACGTGC TACAATGGCG CGTACAGAGG GCTGCAAGCT 1260

099 AGCGATAGTG AGCGAATCCC AAAAAGCGCG TCGTAGTCCG GATCGGAGTC TGCAACTCGA 1320

099 CTCCGTGAAG TCGGAATCGC TAGTAATCGC AAATCAGAAT GTTGCGGTGA ATACGTTCCC 1380

099 GGGCCTTGTA CACACCGCCC GTCACACCAT GGGAGTGGGT TGCACCAGAA GTAGATAGCT 1440

099 TAACCTCCGG GAGGGCGTTT ACCACGGTGT GATTCATGAC TGGGGTGAAG TCGTAACAAG 1500

099 GTAACCCTAG GGGAACCTGG GGTTGGATCA CCTCCTTTAT G 1541

Figure 4.6 – Nucleotide sequence of 16S rDNA of A. hydrophila 099

161

GGGACGCAAA TTCAGCTTGT GTAAGCCCCG CTGAAAGCCT GATTTCTTTG AATTCTCTGT 60

TATTCATAGG GTTAGAATCG ATCCTCTCGT GATTTTAAGG ATGTGATATT ACATCACCTC 120

AACAAACCGA TGAGTCTACC CGCACGCTAC CAGAAGTTAG GTTTTAACTG GAAAACTCCG 180

TGTTTCCGTT GACTCTACCC CCGTGTTACA GGGCGGGGGT ATCGGTGCGG CGGTGTACAC 240

AATGCACATC CTGTCCTGCC TTTGATGTCG AAACCGGACA AGATGTGTTT TTGGAGTACC 300

GCCATTCGCG GCAACATCCA AAAACGGCTC ATCAGGGCGA TGGCCTCACT GCGTGAACCA 360

TCACCCGTTA GCAGTGTTAT TTCTCCGGCG TGTCGTCAAT CACTAAATCG GAACCCTGAA 420

AGGGAACCCC CGATTTAGCG TTTGACGAGT TCAGACCACA GAGATGCGGA TGAATATCCA 480

GAAAAGACGC CTCCAAATTC GCGCTCAGAA GCTCTATGAG GGGATGAAGG TCATCATCAA 540

GGGGATGGAG TGAGGCAAAC GCCGTAAATG CCTCTACGGC GTTCTGGTGA CGTGTAATCA 600

TGGCAAGCAT GGTAGGATTC GTGTCAGTCA TGGAGACCTC CGTAACAGGT TTTCGTGATA 660

GCAGGGGGCG GGAACTCGTA ATTCCTTCCC CCTGCGCCCA TCAGATCATA ATTCCGCTAC 720

ACGGGCAAGC CGCCACGGGA AGGGTGCACT CCTCGTGGGA ACTCGGTGCG CTCCCTTCCC 780

GTGCCCGCCT GCCAGCTCGT GCCATTCTGA CCCGTCCGGC ACAGACAGCC AGAAACCACG 840

CGAGAGAGTC CGCACCGCTC GCCGCAAGAC GTGATTAGCG AGCAACGCGA GTCTGTCACG 900

TCGAGGAAGC GGAATATCAC CTGAACGGGC ATAGAAAACC GTACGCACAG TACCCACAGA 960

AAAACACGAT GCAGGATAGG GCAAACGCCG CAAAATGCTA GCATTTTACT CGTTTGCCCG 1020

GTGACCTACA GCAGTTTCCA GAAGTGATGT TTTCGTGGCT GGCATCGCGC ACAGAGTTAC 1080

AAGATACAGA AGGGAACCTC CCGCAAGCCG CGCCACAAGC GGAATCATTT GACAGATAGT 1140

TATGTTCAGG CATAACTTAG TTATGTTCAG GCATAACTAT TTAAGGAATT TTCATGCGAA 1200

AAGTCACACA GGTTGACCTC GAAACCGGAG AGGATTTGGG CGGGTTTGTC GCCGTGATCC 1260

GTCCCAAGCA AAAATCATCG TTCGAGAGGC ATTTCACTAT GAATCAGGCA GCACTCAAAA 1320

TCATCGCTAC AGAACTGAAC CATGAGCAGA CAAAAGTACT CATGATGCTT CTCGCAGACC 1380

TGGACTACGA AAATTACATT CAGGTGGCAC AAATCGACAT TGCAGAATCA TTGGGAATGA 1440

AAAAACCAAA TGTTAGCAAA GCTGTTAAAA ACCTGATTGA GTTCGGAATA ATCCTTGAGG 1500

GGCCAAAGAT AGGCCGAAGC AAAACCTACC GCCTGAACCC TCAGTTCGGC TGGAAAGGCA 1560

CGGTAAGCAA CCACAAAAAA GCACTCAAAA ACGGCCTCAG TATCATTCAG GGTGGCAAGG 1620

TGTGAGCCGT ACCATCTTGG TACCCTTCCG ATCCAAACCT CCCTTTCATT TCACGATTTA 1680

AGCGGCTTTT CGCCCCGCTC CAGTGGGTCA GTTCATGCAA GCCGGTAGCG TAAAAATTTG 1740

CGGCATCGGC AAACAAATGA CGCTCAGGCA ACCAGATTTC ATCCGTTAGC GGTTTAAAAA 1800

ATGCCTGCTG GCCACGTTCT GTAATATTCG CTCCTGTAAG CCGAATTCCA GCACACT 1857

Figure 4.7 – Nucleotide sequence of pMLM extracted from H. alvei 068

162

halI/halR H.alvei AF503776 GTACTTAAGT ACACCGCTGC CGCCGAGACT GTAGAACAAA CTTCAGGGAT GCCATATGTT 60

halI/halR068 ---------- ---------- ---------- ---------- CTTCAGGGAT GCCATATGTT 20

halI/halR067 ---------- ---------- ---------- ---------- CTTCAGGGAT GCCATATGTT 20

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus .......... .......... .......... .......... CTTCAGGGAT GCCATATGTT

halI/halR H.alvei AF503776 TTCTATTTTC AATAAAAATC AGATAATAAC GGAAACGCTT CGTGATTATA TCGATAGAAA 120

halI/halR068 TTCTATTTTC AATAAAAATC AGATAATAAC GGAAACGCTT CGTGATTATA TCGATAGAAA 80

halI/halR067 TTCTATTTTC AATAAAAATC AGATAATAAC GGAAACGCTT CGTGATTATA TCGATAGAAA 80

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus TTCTATTTTC AATAAAAATC AGATAATAAC GGAAACGCTT CGTGATTATA TCGATAGAAA

halI/halR H.alvei AF503776 ACTGTCCCAG TTTGGTAGCC CTGAGTACGC TTACACCGTC GTCAATAAGA AAAACCCGTC 180

halI/halR068 ACTGTCCCAG TTTGGTAGCC CTGAGTACGC TTACACCGTC GTCAATAAGA AAAACCCGTC 140

halI/halR067 ACTGTCCCAG TTTGGTAGCC CTGAGTACGC TTACACCGTC GTCAATAAGA AAAACCCGTC 140

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus ACTGTCCCAG TTTGGTAGCC CTGAGTACGC TTACACCGTC GTCAATAAGA AAAACCCGTC

halI/halR H.alvei AF503776 AAAACTGCTT ATCATCTCAA GCTATCCTGA TGAATGGGTA AACCTGTACA TTGCGAATAA 240

halI/halR068 AAAACTGCTT ATCATCTCAA GCTATCCTGA TGAATGGGTA AACCTGTACA TTGCGAATAA 200

halI/halR067 AAAACTGCTT ATCATCTCAA GCTATCCTGA TGAATGGGTA AACCTGTACA TTGCGAATAA 200

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus AAAACTGCTT ATCATCTCAA GCTATCCTGA TGAATGGGTA AACCTGTACA TTGCGAATAA

halI/halR H.alvei AF503776 CCTGCAGCAC ATTGACCCGG TGATCCTGAC CGCGTTTAAA CGCACGTCCC CTTTCGTGTG 300

halI/halR068 CCTGCAGCAC ATTGACCCGG TGATCCTGAC CGCGTTTAAA CGCACGTCCC CTTTCGTGTG 260

halI/halR067 CCTGCAGCAC ATTGACCCGG TGATCCTGAC CGCGTTTAAA CGCACGTCCC CTTTCGTGTG 260

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus CCTGCAGCAC ATTGACCCGG TGATCCTGAC CGCGTTTAAA CGCACGTCCC CTTTCGTGTG

halI/halR H.alvei AF503776

GGATGAGAAC ATCACGTTGA TGTCTGACCT CAAGATCTCA AAGATTTTCT CTTTATCCAA 360

halI/halR068 GGATGAGAAC ATCACGTTGA TGTCTGACCT CAAGGTCTCA AAGATTTTCT CTTTATCCAA 320

halI/halR067 GGATGAGAAC ATCACGTTGA TGTCTGACCT CAAGGTCTCA AAGATTTTCT CTTTATCCAA 320

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus

GGATGAGAAC ATCACGTTGA TGTCTGACCT CAAG.TCTCA AAGATTTTCT CTTTATCCAA

halI/halR H.alvei AF503776 GAAATACAAC ATCGCCAACG GCTATACTTT CGTCCTGCAC GATCATCTCA ACAATCTGGC 420

halI/halR068 GAAATACAAC ATCGCCAACG GCTATACTTT CGTCCTGCAC GATCATCTCA ACAATCTGGC 380

halI/halR067 GAAATACAAC ATCGCCAACG GCTATACTTT CGTCCTGCAC GATCATCTCA ACAATCTGGC 380

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus GAAATACAAC ATCGCCAACG GCTATACTTT CGTCCTGCAC GATCATCTCA ACAATCTGGC

halI/halR H.alvei AF503776 ACTACTATCA TTAATTATTG ATAGCAATAT GAAAGCGAAT CTGGAAGAGC AGTTCTCTTC 480

halI/halR068 ACTACTATCA TTAATTATTG ATAGCAATAT GAAAGCGAAT CTGGAAGAGC AGTTCTCTTC 440

halI/halR067 ACTACTATCA TTAATTATTG ATAGCAATAT GAAAGCGAAT CTGGAAGAGC AGTTCTCTTC 440

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus ACTACTATCA TTAATTATTG ATAGCAATAT GAAAGCGAAT CTGGAAGAGC AGTTCTCTTC

halI/halR H.alvei AF503776 AGAGAAAGGC AACTTACAGA TGTTACTCAT TGAGATTAAT GAGCAAATGT ATCGGCTCGT 540

halI/halR068

AGAGAAAGGC AACTTACAGA TGTTACTCAT TGAGATTAAT GAGCAAATGT GTCGGCTCGT 500

halI/halR067 AGAGAAAGGC AACTTACAGA TGTTACTCAT TGAGATTAAT GAGCAAATGT ATCGGCTCGT 500

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus AGAGAAAGGC AACTTACAGA TGTTACTCAT TGAGATTAAT GAGCAAATGT .TCGGCTCGT

halI/halR H.alvei AF503776

GCAGTCAGTT TCGGAAGATA AGGATGGTTC AGAGATGGGC GTAAGCAAAG CAACGTTTAC 600

halI/halR068 GCAGTCAGTT TCGGTAGATA AGGATGGTTC TGAGATGGGC GTAAGCAAAG CAACGTTTAC 560

halI/halR067 GCAGTCAGTT TCGGTAGATA AGGATGGTTC TGAGATGGGC GTAAGCAAAG CAACGTTTAC 560

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus

GCAGTCAGTT TCGG.AGATA AGGATGGTTC .GAGATGGGC GTAAGCAAAG CAACGTTTAC

halI/halR H.alvei AF503776

AGCCAGAGAA CATGAAGTAC TTTACTGGGC GAGTATGGGG AATACTTACG CGGAGATCGC 660

halI/halR068 AGCCAGAGAA CATGAAGTAC TTTACTGGGC GAGTATGGGG AAAACTTACG CGGAGATCGC 620

halI/halR067 AGCCAGAGAA CATGAAGTAC TTTACTGGGC GAGTATGGGG AAAACTTACG CGGAGATCGC 620

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus

AGCCAGAGAA CATGAAGTAC TTTACTGGGC GAGTATGGGG AA.ACTTACG CGGAGATCGC

halI/halR H.alvei AF503776

CACAATCATT GGTATTTCAG TAAGAACGGT TAAATTTCAC ATGGGCAACG TGGTAAGTAA 720

halI/halR068 CACAATCATT GGGATTTCAG TAAGAACGGT TAAATTTCAC ATGGGCAACG TGGTAAGTAA 680

halI/halR067

CACAATCATT GAGATTTCAG TAAGAACGGT TAAATTTCAC ATGGGCAACG TGGTAAGTAA 680

halI059 ---------- ---------- ---------- ---------- ---------- ---------- 0

halI071 ---------- ---------- ---------- ---------- ---------- ---------- 0

Clustal Consensus

CACAATCATT G..ATTTCAG TAAGAACGGT TAAATTTCAC ATGGGCAACG TGGTAAGTAA

Figure 4.8 - Multiple sequence alignment of halI gene of H. alvei 059, 068, 071, and

Enterobacter sp. 067 (this study) with halI gene of H. alvei (Genbank

accession number AF503776). The differences of identity are indicated

by gray shading.

163

halI/halR H.alvei AF503776 ATTGGGTGTG AGTAACGCCC GTCAGGCGAT CAGGCTGGGC GTTGAACTCG AACTGATTAC 780

halI/halR068 ATTGGGTGTG AGTAACGCCC GTCAGGCGAT CAGGCTGGGC GTTGAACTCG AACTGATTAC 740

halI/halR067 ATTGGGTGTG AGTAACGCCC GTCAGGCGAT CAGGCTGGGC GTTGAACTCG AACTGATTAC 740

halI059 ---------- ---------- ---------- ---------- ---------- AACTGATTAC 10

halI071 ---------- ---------- ---------- ---------- ---------- AACTGATTAC 10

Clustal Consensus ATTGGGTGTG AGTAACGCCC GTCAGGCGAT CAGGCTGGGC GTTGAACTCG AACTGATTAC

halI/halR H.alvei AF503776 ACCAATGCAG TCTTAATTAC CGCCACTGAC ATCGGCCAGT GCTCAAATTC AGGCAGTGAA 840

halI/halR068 ACCAATGCAG TCTTAATTAC CGCCACTGAC ATCGGCCAGT GCTCAAATTC AGGCAGTGAA 800

halI/halR067 ACCAATGCAG TCTTAATTAC CGCCACTGAC ATCGGCCAGT GCTCAAATTC AGGCAGTGAA 800

halI059

ACCAATGCAG TCGTAA---C GGCTATCGAC ATTGGCCAAT TAACCAGCTG GGACTGAATA 67

halI071 ACCAATGCAG TCTTAATTAC CGCCACTGAC ATCGGCCAGT GCTCAAATTC AGGCAGTGAA 70

Clustal Consensus

ACCAATGCAG TC.TAATTAC .GC.A..GAC AT.GGCCA.T ...C.A..T. .G.C.G...A

halI/halR H.alvei AF503776 CAACCTTCAA ATGCATTAAT TTTTGTCGCC ATTTTCGCCT GACTATCAGA GTCCGTAGGC 900

halI/halR068 CAACCTTCAA ATGCATTAAT TTTTGTCGCC ATTTTCGCCT GACTATCAGA GTCCGTAGGC 860

halI/halR067 CAACCTTCAA ATGCATTAAT TTTTGTCGCC ATTTTCGCCT GACTATCAGA GTCCGTAGGC 860

halI059

CCACCGACTG AAGCGGTTAT TTTGGCTGCC ATTTTATTCT GGCTGACAGA GTCTGTCGGG 127

halI071 CAACCTTCAA ATGCATTAAT TTTTGTCGCC ATTTTCGCCT GACTATCAGA GTCCGTAGGC 130

Clustal Consensus

C.ACC..C.. A.GC..T.AT TTT.G..GCC ATTTT...CT G.CT..CAGA GTC.GT.GG.

halI/halR H.alvei AF503776 AAATAGAGAA GATAAATCCT TTCCTCCTCA CTCAAATATG CCTCTTTAAG TACCCTCACC 960

halI/halR068 AAATAGAGAA GATAAATCCT TTCCTCCTCA CTCAAATATG CCTCTTTAAG TACCCTCACC 920

halI/halR067 AAATAGAGAA GATAAATCCT TTCCTCCTCA CTCAAATATG CCTCTTTAAG TACCCTCACC 920

halI059

AGATAAACCA GATAAATTCG CTCATCCTCA CTCAGATATG CTTCCTTAAG CGGTTTAATT 187

halI071 AAATAGAGAA GATAAATCCT TTCCTCCTCA CTCAAATATG CCTCTTTAAG TACCCTCACC 190

Clustal Consensus

A.ATA.A..A GATAAAT.C. .TC.TCCTCA CTCA.ATATG C.TC.TTAAG .....T.A..

halI/halR H.alvei AF503776 TGCCAGCCAC TGCGCTTTAG TATTGTCAGC ATAGCGCGGC TAACAATCGT ATAAATGCCG 1020

halI/halR068 TGCCAGCCAC TGCGCTTTAG TATTGTCAGC ATAGTGCGGC TAACAATCGT ATAAATGCCG 980

halI/halR067 TGCCAGCCAC TGCGCTTTAG TATTGTCAGC ATAGCGCGGC TAACAATCGT ATAAATGCCG 980

halI059

TGCCAACCTG AACGTTTCAA TATTGTCAGC ATTGCTCGGC TGACGATAGT GTAAATACCG 247

halI071 TGCCAGCCAC TGCGCTTTAG TATTGTCAGC ATAGCGCGGC TAACAATCGT ATAAATGCCG 250

Clustal Consensus

TGCCA.CC.. ..CG.TT.A. TATTGTCAGC AT.G..CGGC T.AC.AT.GT .TAAAT.CCG

halI/halR H.alvei AF503776 TTTAAGTTAT ATTGACGAGC GTAGTTGATC ATCGCCAGAA AAAGAACCTG ACTAACAGGA 1080

halI/halR068 TTTAAGTTAT ATTGACGAGC GTAGTTGATC ATCGCCAGAA AAAGAACCTG ACTAACAGGA 1040

halI/halR067 TTTAAGTTAT ATTGACGAGC GTAGTTGATC ATCGCCAGAA AAAGAACCTG ACTAACAGGA 1040

halI059

TTGCAACCAT AGTGCCTAGC ATAATTTATC ATCGCTAAAA ATAGTACTTG GCTTAGCGGA 307

halI071 TTTAAGTTAT ATTGACGAGC GTAGTTGATC ATCGCCAGAA AAAGAACCTG ACTAACAGGA 310

Clustal Consensus

TT..A...AT A.TG.C.AGC .TA.TT.ATC ATCGC.A.AA A.AG.AC.TG .CT.A..GGA

halI/halR H.alvei AF503776 TATCTCTCCC CCAGCAAATC TCGAGCGCGA CTTTTGTCGA CAAAGAATCG GCTTGATTCC 1140

halI/halR068 TATCTCTCCC CCAGCAAATC TCGAGCGCGA CTTTTGTCGA CAAAGAATCG GCTTGATTCC 1100

halI/halR067 TATCTCTCCC CCAGCAAATC TCGAGCGCGA CTTTTGTCGA CAAAGAATCG GCTTGATTCC 1100

halI059

TATCGCTCGC CTAACAAATC CCTCGCGCGT GATTTATCAA CAAAAAATCG GCTTGATTCT 367

halI071 TATCTCTCCC CCAGCAAATC TCGAGCGCGA CTTTTGTCGA CAAAGAATCG GCTTGATTCC 370

Clustal Consensus

TATC.CTC.C C.A.CAAATC .C..GCGCG. ..TTT.TC.A CAAA.AATCG GCTTGATTC.

halI/halR H.alvei AF503776 ACTTCACCGG CTGGCAATGA GACATCATGA AAACAAGAGT GGAAGGTATG AGTAATCATA 1200

halI/halR068 ACTTCACCGG CTGGCAATGA GACATCATGA AAACAAGAGT GGAAGGTATG AGTAATCATA 1160

halI/halR067

ACTTCACCGG CTGGCAATGA GACATCGTGA AAACAAGAGT GGAAGGTATG AGTAATCATA 1160

halI059

GTTTCACCGG CAGGCAAAGG CACATCCGAA AAACAAGCCT GAAACGTGTG AGTAATCATA 427

halI071 ACTTCACCGG CTGGCAATGA GACATCATGA AAACAAGAGT GGAAGGTATG AGTAATCATA 430

Clustal Consensus

..TTCACCGG C.GGCAA.G. .ACATC...A AAACAAG..T G.AA.GT.TG AGTAATCATA

halI/halR H.alvei AF503776 TTTGGCAGTT CGAGCGGCAC AAAACGAACA CTGCAAATCA ACTGCCCTTC ATACAGACCC 1260

halI/halR068 TTTGGCAGTT CGAGCGGCAC AAAACGAACA CTGCAAATCA ACTGCCCTTC ATACAGACCC 1220

halI/halR067 TTTGGCAGTT CGAGCGGCAC AAAACGAACA CTGCAAATCA ACTGCCCTTC ATACAGACCC 1220

halI059

TTAGGCTCAT CCAATGGAAC AAAGCGCACG CTGCAGACCA GCTGTCCCTC ATATAAACCT 487

halI071 TTTGGCAGTT CGAGCGGCAC AAAACGAACA CTGCAAATCA ACTGCCCTTC ATACAGACCC 490

Clustal Consensus

TT.GGC...T C.A..GG.AC AAA.CG.AC. CTGCA.A.CA .CTG.CC.TC ATA.A.ACC.

halI/halR H.alvei AF503776 AAGATATAGC GCGTGTTTGG ATTATCAAAC TCATCAAACT CCATACCGCG GTTGCAAACG 1320

halI/halR068 AAGATATAGC GCGTGTTTGG ATTATCAAAC TCATCAAACT CCATACCGCG GTTGCAAACG 1280

halI/halR067 AAGATATAGC GCGTGTTTGG ATTATCAAAC TCATCAAACT CCATACCGCG GTTGCAAACG 1280

halI059

AGGATGTAAC GTGTATTAGG ATTATCAAAC TCGTCAAACT CCATGTCTTT GTTGCAGACA 547

halI071 AAGATATAGC GCGTGTTTGG ATTATCAAAC TCATCAAACT CCATACCGCG GTTGCAAACG 550

Clustal Consensus

A.GAT.TA.C G.GT.TT.GG ATTATCAAAC TC.TCAAACT CCAT..C... GTTGCA.AC.

halI/halR H.alvei AF503776 ACATCCCAAC CTAGTCGATC GCTGAAAGTT TTTTTTCTCA GTCGATAAAG TTCATCCGAA 1380

halI/halR068 ACATCCCAAC CTAGTCGATC GCTGAAAGTT TTTTTTCTCA GTCGATAAAG TTCATCCGAA 1340

halI/halR067 ACATCCCAAC CTAGTCGATC GCTGAAAGTT TTTTTTCTCA GTCGATAAAG TTCATCCGAA 1340

halI059

ACATCCCATC CCAACCGGTC ACTGAACGTT TTTTTTCTCA GTCGATAGAG TTCATCAGAA 607

halI071 ACATCCCAAC CTAGTCGATC GCTGAAAGTT TTTTTTCTCA GTCGATAAAG TTCATCCGAA 610

Clustal Consensus

ACATCCCA.C C.A..CG.TC .CTGAA.GTT TTTTTTCTCA GTCGATA.AG TTCATC.GAA

halI/halR H.alvei AF503776 CGCACTCCAT TTAGTTCATC ATAACTGACA TCAAATAGTT CAAGCATTCC ATCACCTTGA 1440

halI/halR068 CGCACTCCAT TTAGTTCATC ATAACTGACA TCAAATAGTT CAAGCATTCC ---------- 1390

halI/halR067 CGCACTCCAT TTAGTTCATC ATAACTGACA TCAAATAGTT CAAGCATTCC ---------- 1390

halI059

CGGACCCCAT TCAGTTCTTC GTAACTGACA TCAAATAGTT CAAGCATTCC ---------- 657

halI071 CGCACTCCAT TTAGTTCATC ATAACTGACA TCAAATAGTT CAAGCATTCC ---------- 660

Clustal Consensus

CG.AC.CCAT T.AGTTC.TC .TAACTGACA TCAAATAGTT CAAGCATTCC ATCACCTTGA

Figure 4.8 (continued).

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