Regulation of β-1,6-N-acetyl-D-glucosamineProduction and Escherichia coli Biofilm Formation Public

Abstract

Bacterial biofilms are important for persistence of microbes in the environment or within a host. Insight into the regulatory elements involved in biofilm formation is essential to understand and combat biofilm-associated diseases. In Escherichia coli, the pgaABCD locus is required for production of polymeric β-1,6-N-acetyl-D-glucosamine (PGA), which promotes biofilm formation. The CsrA (carbon storage regulator) RNA-binding protein represses PGA production. However, at the onset of this project further insights into the regulatory elements that influence PGA accumulation were lacking.

Disruption of nhaR, encoding a transcriptional regulator important for survival of E. coli under conditions of high salinity and alkaline pH, was found to greatly reduce biofilm formation without impairing growth. NhaR affected pga expression in response to increasing monovalent cation concentrations in the medium and to alkaline pH. pgaA transcript levels were reduced 200-fold and PGA was undetectable upon disruption of nhaR. Purified NhaR-His6 bound specifically to the pgaA promoter region and was necessary for in vitro transcription. Recent studies by other groups suggest that induction of nhaR occurs during colonization of the gut and PGA may be critical for this process.

A biofilm screen of a panel of single-gene deletions of GGDEF and/or EAL domain proteins, responsible for synthesis and degradation of the secondary messenger c-di-GMP, respectively, indicated that several of these genes affect biofilm formation. Over-expression of YhjH (EAL domain) decreased biofilm and PGA accumulation, whereas expression of YdeH (GGDEF) from a plasmid increased both. Since these two proteins have been shown to affect intracellular c-di-GMP levels, these results suggested that c-di-GMP modulates PGA synthesis. Nevertheless, over-expression of YdeH or YhjH did not affect a pgaA'-‘lacZ translational fusion, suggesting that c-di-GMP does not alter transcription-translation of pgaA. We developed an in vitro PGA synthesis assay using membrane preparations; however addition of c-di-GMP failed to enhance in vitro PGA synthesis. Membranes from strains depleted for c-di-GMP (over-expressing yhjH) were unable to synthesize PGA even in the presence of added c-di-GMP. These results may suggest the existence of an unidentified c-di-GMP responsive factor or the requirement of c-di-GMP for a functional Pga complex. These studies present additional regulatory elements that modulate PGA expression and biofilm formation in response to growth conditions.

Table of Contents

Chapter 1. Introduction.....................................................................................................1

Rationale and specific aims .....................................................................................1

Background and significance ...................................................................................2

Biofilms ........................................................................................................2

Biofilm history ..............................................................................................3

Biofilms in the environment .........................................................................4

Biofilms in industry and medicine................................................................5

Biofilm structure and development ..........................................................................7

Surface structures involved in biofilm formation ....................................................8

Protein..........................................................................................................9

Polysaccharides .........................................................................................11

Regulation of biofilm formation ............................................................................12

Environmental factors ................................................................................12

Signal transduction and quorum sensing ...................................................14

Catabolite repression ................................................................................. 15

Csr system ..................................................................................................15

c-di-GMP ...................................................................................................17

Conclusion .............................................................................................................17

References ..............................................................................................................19

Chapter 2. Environmental Influences on Biofilm Development. ................................37

Summary ................................................................................................................38

Abstract ..................................................................................................................39

Introduction ............................................................................................................39

Surface factors and hydrodynamic effects .............................................................40

Approach and initial attachment to the surface .....................................................42

Motility and chemotaxis .............................................................................42

Surface sensing? ........................................................................................46

Environmental effects on surface attachment proteins ..............................47

Curli ...........................................................................................................47

Type 1 fimbriae ..........................................................................................50

Antigen 43 and related proteins .................................................................51

Conversion from temporary to permanent attachment ..........................................52

Environmental effects on matrix polysaccharides .................................................54

Conditions and factors mediating biofilm dispersal ..............................................58

Mixed species biofilms ..........................................................................................62

Conclusions and outlook ........................................................................................64

References ..............................................................................................................66

Chapter 3. The Cation-responsive Protein NhaR of Escherichia coli Activates pgaABCD Transcription, Required for Production of the Biofilm Adhesin Poly-β-1,6-N-acetyl-D-glucosamine....................................................................99

Summary ..............................................................................................................100

Abstract ................................................................................................................101

Introduction ..........................................................................................................101

Materials and Methods .........................................................................................105

Results and Discussion ........................................................................................115

Conclusions ..........................................................................................................123

Acknowledgements ..............................................................................................124

References ............................................................................................................125

Chapter 4. Regulation of PGA accumulation and biofilm formation in Escherichia coli by GGDEF and EAL domain proteins...................................................149

Summary ..............................................................................................................150

Abstract ................................................................................................................152

Introduction ..........................................................................................................153

Materials and Methods .........................................................................................157

Results ..................................................................................................................162

Discussion ............................................................................................................170

Acknowledgments................................................................................................173

References ............................................................................................................174

Chapter 5. Dissertation Discussion. ..............................................................................196

Role of PGA in biofilm formation .......................................................................196

Elucidating the NhaR regulon ..............................................................................196

Biofilm forming capacities of E. coli isolates from diverse environments ..........199

Investigations into the Pga proteins and PGA synthesis machinery ....................201

Outlook ................................................................................................................202

References ............................................................................................................203

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Microbiology
Mot-clé	Escherichia coli biofilms regulation CsrA NhaR c-di-GMP
Committee Chair / Thesis Advisor	Romeo, Tony, University of Florida
Committee Members	Shafer, William M, Emory University Rather, Phil N, Emory University Moran Jr., Charles, Emory University Scott, June R, Emory University

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