autoinducer-2 (ai-2) mediated quorum sensing in escherichia coli

179 Pages·2004·2.08 MB·English

ABSTRACT
Title of dissertation: AUTOINDUCER-2 (AI-2) MEDIATED QUORUM
SENSING IN ESCHERICHIA COLI
Liang Wang, Doctor of Philosophy, 2004
Dissertation directed by: Professor William E. Bentley, Department of
Chemical Engineering &
Professor Steven W. Hutcheson, Department of
Cell Biology and Molecular Genetics
Bacteria have evolved complex genetic circuits to regulate their physiological
activities and behaviors in response to extracellular signals. In a process termed
“quorum sensing”, or density-dependent gene regulation, bacteria produce, release and
respond to certain signaling molecules termed autoinducers. The bacterial autoinducer-2
(AI-2) has received intense interest recently because the gene for its synthesis, luxS, is
common in a large number of Gram-negative and Gram-positive bacterial species. In
this study, the luxS controlled genes were identified in Escherichia coli K12 strain
under two different growth conditions using DNA microarrays. Deletion of the luxS was
shown to affect expression of genes involved in AI-2 transport (the lsr operon) and
methionine biosynthesis (metE), and to a lesser degree those involved in methyl transfer,
iron uptake, resistance to oxidative stress, utilization of various carbon sources, and
virulence. The effects of glucose on extracellular AI-2 level were investigated further. It
was shown that both AI-2 synthesis and uptake in Escherichia coli are subject to
catabolite repression through the cAMP-CRP complex. This complex directly
stimulates transcription of the lsr (luxS regulated) operon and indirectly represses luxS
expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in
the upstream region of the lsr promoter and works with LsrR repressor to regulate AI-2
uptake. This study, for the first time, has shown that quorum sensing regulates specific
activities in E. coli K12, and has elucidated regulatory mechanisms for AI-2
biosynthesis and transport in this organism. With a better understanding of AI-2/luxS
mediated gene regulation, we may be able to develop strategies for harnessing AI-2
quorum sensing for our advantage in bioreactor studies and ultimately in control of the
bacterial pathogenicity.
AUTOINDUCER-2 (AI-2) MEDIATED QUORUM SENSING IN
ESCHERICHIA COLI
by
Liang Wang
Dissertation submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfillment
of the requirement for the degree of
Doctor of Philosophy
2004
Advisory Committee:
Professor Steven W. Hutcheson, Chair
Professor William E. Bentley, Co-Chair
Associate Professor David C. Straney
Associate Professor Richard C. Stewart
Professor Siba K. Samal (Dean’s Representative)
© Copyright by
Liang Wang
2004
Doctor of Philosophy
DEDICATION
I would like to dedicate this work to my wife, Min Zhou, without whom none of
the following would have been possible.
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ACKNOWLEDGEMENTS
I would like to take this opportunity to gratefully acknowledge my advisors, Dr.
William E. Bentley and Dr. Steven W. Hutcheson, for their invaluable time, support,
wisdom, and guidance during all the years of my graduate research. I would also like to
thank Dr. Richard C. Stewart, Dr. David C. Straney, and Dr. Siba K Samal for their
time, suggestions, and encouragement.
I would like to acknowledge F. P. Schwarz for the gift of CRP and B. L. Bassler,
H. Aiba, R. Kolter, A. Hochschild, L. I. Rothfield, and M. Berlyn for generously
providing strains and plasmids used in this study. I would like to thank Mr. Alvaro
Godínez for assistance in the DNA microarray experiments. This research was
supported by the US Army Engineering, Research and Development Center, Edgewood,
MD (Grant #DAAM01-96-0037) and the Center for Biosystems Research, UMBI.
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LIST OF CONTENTS
LIST OF FIGURES..................................................................................................................................V
LIST OF TABLES.................................................................................................................................VII
CHAPTER 1 GENERAL INTRODUCTION..........................................................................................1
WHAT IS QUORUM SENSING?...................................................................................................................1
ACYL-HOMOSERINE LACTONE (AHL) MEDIATED QUORUM SENSING.....................................................2
THE VIBRIO FISCHERILUXI/LUXR SYSTEM..............................................................................................2
LUXI/LUXR TYPE FAMILY AND AHL BIOSYNTHESIS..............................................................................7
SDIA, THE LUXR HOMOLOGUE IN E. COLI..............................................................................................11
QUORUM QUENCHING............................................................................................................................20
PEPTIDE MEDIATED QUORUM SENSING..................................................................................................20
PARALLEL QUORUM SENSING SYSTEMS IN VIBRIO HARVEYI...................................................................21
AI-2 SYSTEM AND LUXS FAMILY...........................................................................................................27
BIOSYNTHESIS AND FORMATION OF AI-2 MOLECULES...........................................................................28
AI-2 AND LUXSCONTROLLED BEHAVIORS..............................................................................................32
STATEMENT OF PURPOSE........................................................................................................................35
CHAPTER 2 CAMP AND CAMP RECEPTOR (CRP) INFLUENCE BOTH SYNTHESIS AND
UPTAKE OF EXTRACELLULAR AUTOINDUCER-2 INESCHERICHIA COLI.........................37
ABSTRACT..............................................................................................................................................37
INTRODUCTION.......................................................................................................................................37
MATERIALS AND METHODS....................................................................................................................42
RESULTS.................................................................................................................................................49
DISCUSSION............................................................................................................................................73
CHAPTER 3 GENOMIC EXPRESSION PROFILING REVEALED GROWTH CONDITION
DEPENDENT GENE REGULATION BY LUXS IN ESCHERICHIA COLI K12.............................82
ABSTRACT..............................................................................................................................................82
INTRODUCTION.......................................................................................................................................83
MATERIALS AND METHODS....................................................................................................................86
RESULTS AND DISCUSSION.....................................................................................................................89
CONCLUSION........................................................................................................................................112
CHAPTER 4 GENOMIC EXPRESSION PROFILING OF ESCHERICHIA COLI GROWN ON
GLUCOSE..............................................................................................................................................115
ABSTRACT............................................................................................................................................115
INTRODUCTION.....................................................................................................................................115
MATERIALS AND METHODS..................................................................................................................117
RESULTS AND DISCUSSION...................................................................................................................120
CONCLUSION........................................................................................................................................144
CHAPTER 5 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS....................................146
REFERENCES.......................................................................................................................................151
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LIST OF FIGURES
Figure 1-1 The LuxR/LuxI-based quorum sensing in Vibrio fisheri................................5
Figure 1-2 Biosynthesis of the acyl-homoserine lactone (AI-1).....................................12
Figure 1-3 Structures of several N-acyl homoserine lactone molecules.........................14
Figure 1-4 SdiA mediated gene regulation in E. coli.....................................................17
Figure 1-5 Peptide mediated quorum sensing in Gram-positive bacteria.......................22
Figure 1-6 Three parallel quorum sensing systems in Vibrio harveyi............................24
Figure 1-7 Biosynthesis and formation of AI-2 signaling molecules.............................29
Figure 2-1 Pathways for AI-2 biosynthesis and SAM utilization...................................40
Figure 2-2 Effects of glucose on extracellular AI-2 activity..........................................50
Figure 2-3 crp and cya deletions increase extracellular AI-2 activity............................53
Figure 2-4 Effects of cAMP and CRP on the transcription of luxS and pfs ...................56
Figure 2-5 No binding of cAMP-CRP to luxS promoter region.....................................58
Figure 2-6 AI-2 activity profiles of E. coli lsr mutants..................................................61
Figure 2-7 Regulation of the transcription of E. coli lsr operon.....................................64
Figure 2-8 cAMP-CRP binds to upstream region of the lsr promoter............................69
Figure 2-9 (cid:1)s negatively regulates expression of the lsr operon.....................................71
Figure 2-10 Conceptual model AI-2 synthesis and uptake in E. coli.............................78
Figure 3-1 Growth of wild type and (cid:2)luxS mutant of the E. coli W3110......................91
Figure 3-2 Swimming motility is not affected by luxS deletion.....................................93
Figure 3-3 Biofilm formation is not affected by luxS deletion.......................................95
Figure 3-4 Transcriptional organization of the lsr operon region.................................101
Figure 3-5 Deletion of luxS affects methionine metabolism........................................107
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Figure 4-1 Effects of glucose on growth of the E. coli cells.........................................121
Figure 4-2 The expression of central metabolic pathway components in response to the
presence of glucose...............................................................................................125
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LIST OF TABLES
Table 1-1 LuxI/LuxR Type Family...................................................................................8
Table 2-1 Bacterial strains and plasmids used in this study...........................................43
Table 2-2 Oligonucleotide primers used in this study....................................................45
Table 2-3 Regulation of the expression of lsrACDBFG operon .....................................66
Table 3-1 Effects of luxS deletion on gene expression patterns under two growth
conditions................................................................................................................99
Table 3-2 Genes regulated by luxS at OD 2.4 in LB....................................................103
Table 3-3 Genes regulated by luxS at OD 1.0 in LB plus glucose................................111
Table 4-1 Functional categories of the differentially expressed genes.........................123
Table 4-2 Effects of carbon sources on expression of several genes............................129
Table 4-3 Differentially expressed genes involved in nitrogen metabolism and
biosynthesis...........................................................................................................130
Table 4-4 Differentially expressed transport genes......................................................135
Table 4-5 Differentially expressed genes encoding regulatory proteins......................138
Table 4-6 Differentially expressed genes involved in cell envelope and cellular
processes...............................................................................................................142
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