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Regulation of Starvation and Nonculturability in The REGULATION OF STARVATION AND NONCULTURABILITY IN THE MARINE PATHOGEN, Vibrio vulnificus S. Diane McDougald A thesis submitted for the degree of Doctor of Philosophy School of Microbiology and Immunology University of New South Wales Sydney, Australia August 2000 ACKNOWLEDGEMENTS First I would like to express my sincere appreciation to my supervisor, Staffan Kjelleberg, for always having a positive attitude, even during my worst failures, and for always having time for discussions. I also would like to thank Staffan for his gently way of guidance and for letting me follow my ideas, no matter how wild. I would also like to thank Dr. Jim Oliver, whose enthusiasm in this project inspired me to pursue a Ph.D. in the first place. I look forward to continued collaboration; thank you for teaching me to be a scientist. Also, I would like to thank Michael Givskov and Lone Gram for their helpful discussions and encouragement. Special thanks go to Christine Paludin-Muller, Dieter Weichart, Daniel Tillett and Sujatha Srinivasan for collaborations on this project. Thanks Dieter for companionship and help during all those long days and nights and all those 2D gels! I especially thank Sujatha who has been down the long road with me. Working with you on this project has certainly made it more enjoyable. I would like to thank all the members of the SK group and CMBB for all their companionship and for making the lab an enjoyable place to work. For their help with sanity-inducing maintenance of cognition (SIMC), a VERY special thanks goes to "The Girls"; Emma Beacham, Katie Crass, Su Egan, Julie Lim, Carolina Tillett, and Hanna Banana for all those times; and the boys, Mike Manefield and Greg Fallon, for all those Friday lunches. I would also like to thank my mother for her many years of support and encouragement, without which I would never have accomplished this. Finally, I would like to thank my husband, Scott Rice for all the help and suggestions with experiments, papers and ideas on this project, as well as for bringing sanity to my life and being my very best friend. TO MY FAMILY “Research is seeing what everyone else has seen and thinking what no one else has thought” Alber Szent-Gyorgyi “The hypothesis outlined in this communication is controversial. Thus, disagreement is expected and welcomed since in the words of Eugene Odum (Odum, E. P., 1977. The emergence of ecology as a new intergrative discipline. Science 195:1289-1293.) disagreement … is certain not only to generate useful knowledge but (also) to promote the art and science of both the experimental and analytical approaches.” Harold Stevenson ABSTRACT Vibrio vulnificus is a model environmental organism exhibiting a classical starvation response during nutrient limitation as well as a non-culturable state when exposed to low temperatures. In addition to these classic global responses, this organism is an opportunistic pathogen that exhibits numerous virulence factors. This organism was chosen as the model organism for the identification of regulators of the viable but nonculturable response (VBNC) and the starvation- induced maintenance of culturability (SIMC) that occurs when cells are starved prior to low temperature incubation. In order to accomplish this, three indirect approaches were used; proteomics, investigation of intercellular signalling pathways and genetic analysis of regulators involved in these responses. Two-dimensional gel electrophoresis was used to identify proteins expressed under conditions that induced SIMC. It was determined that carbon and long- term phosphorus starvation were important in the SIMC response. V. vulnificus was shown to possess genes, luxS and smcR, that are homologues of genes involved in signalling system system 2 in Vibrio harveyi. Signal molecules were produced upon starvation and the entry to stationary phase in V. vulnificus. Furthermore, a null mutation in smcR, a transcriptional regulator was shown to have pleiotropic effects in V. vulnificus, including up-regulation of numerous virulence factors and a defect in starvation survival and development of the SIMC response. We propose that V. vulnificus possesses a signalling system analogous to that of system 2 in V. harveyi, and that this system is involved in the regulation of stationary phase and starvation adaptation in this organism. i TABLE OF CONTENTS 1 GENERAL INTRODUCTION AND LITERATURE REVIEW.............1 1.1 GENERAL INTRODUCTION.............................................................1 1.2 DESCRIPTION OF THE MODEL ORGANISM - Vibrio vulnificus ....2 1.2.1 Historical perspective ...................................................................3 1.2.2 Syndromes of disease caused by V. vulnificus ...............................3 1.2.3 Virulence factors ..........................................................................6 1.2.4 Population heterogeneity...............................................................8 1.2.5 Distribution of V. vulnificus ..........................................................9 1.2.6 Adaptive responses of V. vulnificus.............................................10 1.3 NUTRIENT LIMITATION................................................................13 1.3.1 Ultramicrobacteria......................................................................15 1.3.2 Starvation-induced differentiation...............................................17 1.3.3 Expression of proteins in response to starvation..........................22 1.3.4 Effect of limiting nutrient on starvation response ........................23 1.3.5 Global regulators of starvation response......................................27 1.3.6 Conclusions ................................................................................37 1.4 THE VIABLE BUT NONCULTURABLE RESPONSE.....................38 1.4.1 Historical Perspective .................................................................38 1.4.2 The Viable but Nonculturable State ............................................39 1.4.3 Formation of VBNC cells ...........................................................40 1.4.4 Methods of assessing the VBNC state.........................................41 1.4.5 Physiology of nonculturable cells ...............................................49 1.4.6 Nucleic acids of nonculturable cells............................................50 1.4.7 Potential virulence of VBNC cells ..............................................51 1.4.8 Reports of in vitro resuscitation ..................................................54 1.4.9 Genetics of the VBNC response..................................................59 1.4.10 Conclusions ................................................................................64 1.5 INTERCELLULAR SIGNALLING ...................................................66 1.5.1 Signalling in Gram-positive bacteria...........................................68 1.5.2 Signalling in differentiating Gram-negative bacteria ...................69 ii 1.5.3 Signalling in non-differentiating Gram-negative bacteria ............70 1.5.4 Eucaryotic interactions................................................................78 1.5.5 Conclusions ................................................................................79 2 STARVATION-INDUCED MAINTENANCE OF CULTURABILITY81 2.1 ABSTRACT.......................................................................................81 2.2 INTRODUCTION..............................................................................82 2.3 MATERIALS AND METHODS........................................................83 2.3.1 Bacterial strains and culture conditions .......................................83 2.3.2 Determination of colony forming units .......................................85 2.3.3 Pre-starvation and cold incubation experiments ..........................85 2.3.4 Pulse-labelling of C7184(T) for two-dimensional PAGE ............86 2.3.5 Resolution of pulse-labelled cell proteins of two-dimensional PAGE . ...........................................................................................87 2.4 RESULTS..........................................................................................88 2.4.1 Starvation survival of Vibrio vulnificus C7184(T).......................88 2.4.2 Effect of pre-starvation on culturability at 4°C............................89 2.4.3 Two-dimensional gel analysis of proteins induced after 1 hour of carbon, nitrogen, phosphorus or multiple-nutrient starvation.......97 2.5 DISCUSSION ..................................................................................110 2.5.1 Starvation survival at 24°C .......................................................111 2.5.2 Effects of starvation conditions on culturability at 4°C..............112 2.5.3 Analysis of protein synthesis during starvation .........................116 2.6 ACKNOWLEDGEMENTS..............................................................118 3 MOLECULAR ANALYSIS OF THE VBNC RESPONSE ..................119 3.1 ABSTRACT.....................................................................................119 3.2 INTRODUCTION............................................................................120 3.3 MATERIALS AND METHODS......................................................124 3.3.1 Bacterial stains and plasmids ....................................................124 3.3.2 Media, growth and screening conditions ...................................127 3.3.3 Fixation of bacterial cells..........................................................128 3.3.4 Hybridisation............................................................................129
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