Analysis of the Role of Rhodobacter sphaeroides CrpO in Tolerance to NaCl Susana Retamal A Thesis Submitted to the Graduate College of Bowling Green State University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Committee: Jill Zeilstra-Ryalls, Advisor Raymond Larsen Scott Rogers © 2010 Susana Retamal All Rights Reserved i ABSTRACT Jill Zeilstra-Ryalls, Advisor For organisms such as Rhodobacter sphaeroides osmoadaptation is essential to survival, since it encounters changes in salinity in its natural habitat of brackish waters. Because it is metabolically versatile, it serves as a useful model in examining how regulatory features of osmoadaptation are integrated into the regulatory circuits that provide the cell with the means to switch from one metabolic option to another. Known components of osmoadaptation in R. sphaeroides include the transport or synthesis of compatible compounds and alterations in fatty acid and phospholipid composition of the cell membrane. However, the transcriptional regulation of the genes associated with these adaptations has not been unraveled. The R. sphaeroides crpO gene has been found to increase NaCl tolerance when present in multicopy. Towards evaluating the role of crpO in NaCl tolerance, strains with different levels of functional CrpO protein were constructed and characterized with respect to each component of osmoadaptation. The main findings are (1) crpO is an essential gene, (2) improved NaCl tolerance by increasing crpO gene dosage is not due to altered compatible solute synthesis or transport; rather (3) fatty acid and phospholipid, especially cardiolipin, composition and quantity are altered in cells with multiple copies of crpO. The importance of this work is its contribution towards understanding the regulatory events associated with osmoadaptation in an organism that is capable of many different energy metabolisms including both aerobic and anaerobic respirations and also anoxygenic photosynthesis. Improved knowledge of how this happens in R. sphaeroides has the potential to increase our understanding for other organisms having the same individual and combined metabolic capabilities. ii ACKNOWLEDGMENTS I would like to extend a heartfelt thank you to my advisor, Dr. Jill Zeilstra-Ryalls, for all your encouragement and for the opportunity to work in your laboratory. Your guidance was extremely important throughout this masters program; it enabled me to think for myself and truly learn about and appreciate every aspect of molecular research. My committee members, Dr. Scott Rogers and Dr. Ray Larsen were both wonderful and inspiring in classes and seminars. I am grateful for your help, suggestions and ideas. Dr. Rogers has taught me how to read a scientific paper with a scientist's eye. Dr. Larsen made our summer seminar very interactive and interesting; you always made time to answer questions and shared chemicals and enzymes that I needed. I would like to thank my husband, Henry and son, Ian for their love and support and especially for being so understanding about this period of my life. To my parents, my Mom for coming all the way from Brazil to help me with my son so I could study and work on my thesis and to my deceased Dad, who as a Biologist inspired me to pursue science and research. A special thanks to Dr. Marcia Salazar-Valentine for helping me through the admission’s process and to my great friend, Gesiel Fisch who was incredibly helpful during hard times. To my friend Dr. Marcela d’ Alincourt Salazar for being available for advise, coffee, dinner, and so many qPCR-related questions. To my dear former laboratory colleague and now one of my closest friends, Yana Fedotova, for being there for me in so many ways. To Dr. Maneewan "Joy" Suwansaard, who from the very first day helped me find my way around the lab and finished running so many of my gels while I was in class. To everyone else in the lab, thank you! iii TABLE OF CONTENTS Page ABSTRACT.………………………………………………………………………………. i ACKNOWLEDGEMENTS.……………………………………………………...………... ii LIST OF FIGURES/TABLES.…………………………………………………………….. vi LIST OF ABBREVIATIONS.…………………………………………………………...... viii CHAPTER I: Background information and specific aims………………………………... 1 Introduction……………………………………………………………………………. 1 A. Bacterial osmoregulation……………………..……............................................ 1 B. Rhodobacter sphaeroides.………………………...…………………….............. 2 C. Salt tolerance in Rhodobacter sphaeroides……………….……………………. 3 D. Rsp1275……………………………….......…………………………………….. 8 Specific Aims………………………………………………………………………….. 11 CHAPTER II: Analysis of the relationship between NaCl tolerance and the intracellular concentration of active CrpO .…………………………………….…………….………… 12 Introduction…………………………………………………………………………..... 12 Materials and Methods……………………………………………………………….... 13 Bacterial strains and plasmids and growth conditions..…………………………...... 13 Measurement of culture densities………………………..………………………….. 13 DNA treatment and manipulations.............................................................................. 14 Construction of a vector for engineering a crpO null mutant strain………………… 14 iv Construction of plasmid pCrpO'-BBR.………………………………….………....... 14 Transformations and conjugations…………………................................................... 15 Results…………………………………………………………………………….......... 17 The crpO gene may be essential….……………………………………………….… 17 Construction of a CrpO "knock down" strain……………………………..………… 18 CrpO-mediated NaCl tolerance does not work through altering compatible solute synthesis or transport.………………………………..……………………………… 20 Is the CrpO-mediated increase in NaCl tolerance specific to photoheterotrophic growth?........................................................................................................................ 23 Does CrpO improve tolerance to other osmotic stressors?.......................................... 25 Discussion........................................................................................................................ 25 CHAPTER III: NaCl-mediated changes in membrane lipid composition and the role of CrpO...........................………………………………………….…………………………... 27 Introduction……………………………………………………………………….……. 27 Materials and Methods……………………………………………………………….... 29 Bacterial and plasmids strains and growth conditions………..…………….………. 29 Extraction of polar lipids...………………………………………………….……..... 29 Thin layer chromatography (TLC) analysis...……………………………….…….... 30 Mass Spectrometry…......……………………………………………...….……........ 30 Chemical determination of polar phospholipid concentrations...……...……………. 30 Results………………………………………………………………………….……… 31 Are NaCl-induced changes in polar phospholipid composition of the membrane 31 v due to changes in enzyme levels or activity?…………………...…………………... Does CrpO affect polar phospholipid composition of the membrane?......…………. 33 Total membrane phospholipid concentrations correlate with crpO copy number.…. 35 Identification………………………………………………………………………... 37 Discussion……………………………………………………………………………... 43 CHAPTER IV: Investigation by quantitative PCR of putative crpO-regulated genes?....... 44 Introduction…………………………………………………………………………..... 44 Materials and Methods……………………………………………………….…........... 45 Bacterial strains and plasmids and growth conditions………..………………….…. 45 RNA isolations and quantitative PCR (qPCR)………............................................... 46 Results……………………………………………………………………….……….... 47 Evaluation of the effect of NaCl on transcription of rpoZ, the standard internal control gene for R. sphaeroides 2.41 qPCR studies….……………....………..…..... 47 Investigation of other genes for their suitability as housekeeping genes………………………………………………………………………………… 48 qPCR-based assessment of the effect of NaCl and crpO copy number on rsp2508- 11 transcription.………………………………………………………………...... 50 Are the rsp2508-11 transcript levels highly variable in cells?…….…..….………… 50 Discussion……………………………………………………….….............................. 53 CHAPTER V: Summary and future perspectives…………….….……………………….. 55 REFERENCES……………………………………………………………….…………..... 57 vi LIST OF FIGURES AND TABLES Figure/Table Page Figure 1. Growth of photosynthetic cultures of R. sphaeroides 2.4.1 in the presence and absence of added NaCl and having either the empty vector pRK415 or plasmid p1275-RK with the rsp1275 gene…………………………………….. 8 Figure 2. Models of the R. sphaeroides Fnr-Crp and E. coli Fnr proteins, and known structure of Crp….…..….....…………..….....…………..….....……..…............ 10 Figure 3. Schematic diagram of the construction of the crpO knock-out vector pSR2…………………………………………………........................................ 18 Figure 4. Schematic diagram of plasmid pCrpO'-BBR…..……………………………… 19 Figure 5. Photoheterotrophic growth of R. sphaeroides 2.4.1 with the plasmids, and with the additions of NaCl, indicated………………………………………….. 23 Figure 6. Chemoheterotrophic growth of R. sphaeroides 2.4.1 with the plasmids indicated, in 0% and 3% added NaCl………………….………………………. 24 Figure 7. Photoheterotrophic growth of R. sphaeroides 2.4.1 with the plasmids indicated in the presence of 3% added KCl…………........................................ 25 Figure 8. Proposed phosphatidylglycerol lipids biosynthetic pathway of R. sphaeroides……………………………………………………………………. 28 Figure 9. TLC of phospholipids extracted from equivalent numbers of photoheterotrophically grown R. sphaeroides 2.4.1 that were incubated with or without added NaCl…………..…..………………………………………… 36 Figure 10. Relative concentrations of total phospholipids