The objective of this study is to clarify the relationship between survivability and energy supply by light in the purple photosynthetic bacteria under non-growing conditions

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(1)Relationship between survivability and energy supply in purple photosynthetic bacteria under non-growing conditions. A doctoral dissertation December 2013. Department of Biological Sciences Graduate School of Science and Engineering Tokyo Metropolitan University. Nanako Kanno.

(2) ABSTRACT. In natural environments, bacteria often face changing environmental conditions such as depletion of essential nutrients and then they enter a non-growing state. Bacterial physiology has been investigated mostly in growing cells, and the physiology including the metabolism of non-growing bacteria is not well characterized. Moreover, the effect of energy level on the metabolism of the starved cells and how they use energy for survival were unclear. The objective of this study is to clarify the relationship between survivability and energy supply by light in the purple photosynthetic bacteria under non-growing conditions. Four species of purple non-sulfur photosynthetic bacteria, Rhodopseudomonas palustris CGA009, Rhodospirillum rubrum S1T, Rhodobacter sphaeroides 2.4.1T, Rubrivivax gelatinosus IL144 were used in this study. The purple bacteria were grown photoheterotrophically in a culture medium containing a low concentration of the carbon source, sodium succinate. When the increase in optical density ceased after the exponential growth phase due to the depletion of the carbon source, the culture was defined as being in carbon-starvation conditions. The starved cultures were incubated for 30 days in the starved conditions in the light and dark, and the viability was determined by plate count (CFU). All species of purple bacteria survived longer in the light when compared to the survival in the dark. Decreasing patterns of CFUs in the dark was different depending on species; i.e., the rates of decrease of CFUs in the dark were different ii.

(3) among strains tested. R. palustris CGA009 and R. sphaeroides 2.4.1T showed clearly higher survivability in the dark compared to the other 2 strains. CFUs of R. rubrum S1T and R. gelatinosus IL144 rapidly decreased in the dark and reached 0.007% and 0.3% of the initial values after 6 days of starvation, respectively. ATP levels in the culture of R. palustris CGA009 and R. rubrum S1Twere maintained similarly in the light, while ATP levels were begun to decrease before the initiation of CFU decrease in the dark. Susceptibility to osmotic stress was also determined using the starved cells of the four species. After exposure to 2.0 M sucrose solution, R. palustris CGA009 and R. sphaeroides 2.4.1T maintained high viability, and in contrast, the sucrose stress markedly decreased the viability in R. rubrum S1T and R. gelatinosus IL144; resistance to the sucrose stresses was somewhat parallel to the survivability in the dark among the four species. To understand the effect of energy supply by light on the metabolism of starved cells, the cellular metabolites were determined in the starved cells of R. palustirs CGA009 which were incubated in the light and dark for 5 days after the beginning of starvation. In addition, to find the genes expressed in the starved cells in the light and dark, transcriptome was analyzed using the microarray. Metabolite profile of starved R. palustris CGA009 cells were largely different between the cells incubated in the light and in the dark. In the light, various amino acids were highly accumulated, while metabolites involved in the glycolytic pathway and the TCA cycle were in the low level. It was also observed in the light that many genes related to protein turnover were expressed. On the other hand, the expression of inorganic-ion transporters was more iii.

(4) remarkable in the dark cells. These results suggested that significant rate of macromolecular turnover was maintained in the starved R. palustris CGA009 cells that were with the support of light energy and metabolites related to the carbon metabolism were used for the biosynthesis. Even in the dark cells, significant levels of macromolecule turnover seemed to proceed, but amino acids may be deficient in the cells. These results suggested that energy supply is important for long-term survival in some bacteria under non-growing conditions. In addition, it was suggested that some metabolism in the starved cells is still active and the utilization of ATP supports the macromolecule turnover in the starved cells for the adaptation to the nutrients limiting conditions.. iv.

(5) CONTENTS. ACKNOWLEDGEMENTS. 1. GENERAL INTRODUCTION. 2. CHAPTER I. 5. Light-dependent survivability in purple photosynthetic bacteria under carbon starvation conditions SUMMARY. 6. INTRODUTSION. 7. MATERIALS AND METHODS. 10. RESULTS. 12. DISCUSSION. 19. SUPPLEMENTAL MATERIALS. 22. REFERENCES. 25. CHAPTER II. 29. Species-dependent survivability in purple photosynthetic bacteria under carbon starvation conditions SUMMARY. 30. INTRODUTSION. 31 v.

(6) MATERIALS AND METHODS. 34. RESULTS. 36. DISCUSSION. 44. SUPPLEMENTAL MATERIALS. 48. REFERENCES. 49. CHAPTER III. 53. Effect of light on metabolomic and transcriptomic profile of starved cells in a purple photosynthetic bacterium Rhodopseudomonas palustris SUMMARY. 54. INTRODUTSION. 55. MATERIALS AND METHODS. 57. RESULTS. 63. DISCUSSION. 88. SUPPLEMENTAL MATERIALS. 95. REFERENCES. 135. GENERAL DISCUSSION. 142. vi.

(7) ACKNOWLEDGEMENTS. I would like to express my gratitude to my supervisors, Drs. Katsumi Matsuura and Shin Haruta for introducing me to the deep field of bacterial ecology and physiology. I appreciate the critical discussions by Dr. Junichi Kato. I also greatly thank Dr. Keizo Shimada for many helpful suggestions, discussions and encouragement. My thanks are due to Dr. Atsushi Kouzuma, Tokyo University of Pharmacy and Life Sciences, for technical instruction in the microarray. I am also grateful to Dr. R. Craig Everroad for critical reading of the manuscript. Special thanks are due to all the members of the Environmental Microbiology Laboratory for help and warm encouragement. Finally I wish to thank my family for let me study to the full.. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to SH (24117519) and Japan Society for the Promotion of Science (JSPS) to NK.. 1.

(8) GENERAL INTRODUCTION. Bacteria often face changing environmental conditions such as depletion of essential nutrients and then they enter a non-growing state. While some bacteria are responding to starvation by forming metabolically inactive endospores or cysts, the vast majority of bacterial species were not able to induce the cell differentiation but it has been observed that they survived under starvation conditions for a long period. Although some stationary phase phenomena such as changes in cell shape, gene expression and increased stress resistance (3, 5, 7) have been the focus of intense research, these observations were limited to some bacterial species and the metabolism of non-growing bacteria is not well characterized. Purple non-sulfur photosynthetic bacteria belong to the alpha and beta subgroups of Proteobacteria and they are classified in 20 genera of 5 orders (4). Their major energy metabolism is characterized by anoxygenic heterotrophic photosynthesis and they utilize organic compounds as carbon source for growth. Purple bacteria also have the ability to obtain energy through fermentation, aerobic respiration, and/or anaerobic respiration (2). They are widely distributed in natural environments and are ecologically important since they substantially contribute to carbon, nitrogen, and sulfur cycles on the earth (4). Some reports have discussed the survivability of purple bacteria under starvation conditions (1, 6). In those study, it was reported that carbon-starved cells of Rhodopseudomonas palustris and Rhodospirillum rubrum maintained viability longer in the light than that in the dark. Purple non-sulfur anoxygenic phototrophic 2.

(9) bacteria use photosystem II type reaction center complexes for photosynthesis; it is expected that they can produce ATP by cyclic photophosphorylation even when organic nutrients are lacking. Thus, it is expected that purple photosynthetic bacteria may be one of the useful groups to investigate the effect of cellular energy on bacterial survival. Although energy supply by photosynthesis under illumination seemed to promote survivability in purple bacteria, it was not clear that the effect of light on intracellular metabolism including ATP level in the starved cells, and whether the effect of illumination is common among purple non-sulfur anoxygenic phototrophic bacteria. In this study, I determined the viability and ATP levels of purple non-sulfur photosynthetic bacteria Rhodopseudomonas palustris strain CGA009, Rhodospirillum rubrum strain S1, in a non-growing state under carbon-starvation conditions in the light and dark. In addition, viability of other two species of purple bacteria belonging to different orders was also investigated comparatively; Rhodobacter sphaeroides strain 2.4.1, and Rubrivivax gelatinosus strain IL144. I also analyzed metabolites of the central and related metabolism and transcriptomic phenotype of starved cells under the light and dark in Rhodopseudomonas palustris CGA009, to understand effect of energy supply by light on metabolism of starved cells.. REFERENCES 1.. Breznak, J.A., C.J. Potrikus, N. Pfennig and J.C. Ensign. 1978. Viability and endogenous substrates used during starvation survival of Rhodospirillum rubrum. J. Bacteriol. 134:381-388. 3.

(10) 2.. Imhoff, J.F. 2006. The phototrophic alpha-proteobacteira, p. 41- 64. In M. Dworkin, S. Falkow, E. Rosenberg, K-H. Schleifer, and E. Stackebrandt (ed.), The Prokaryotes, 3rd ed., vol. 5. Springer, New York.. 3.. Kjelleberg, S., N. Albertson, K. Flärdh, L. Holmquist, A. Jouper-Jaan, R. Marouga, J. Ostling, B. Svenblad and D. Weichart. 1993. How do non-differentiating bacteria adapt to starvation? Antonie van Leeuwenhoek. 63:333-341.. 4.. Madigan, M.T. and D.O. Jung. 2009. An overview of purple bacteria: systematics, physiology, and habitats, p. 1- 15. In C.N. Hunter, F. Daldal, M.C. Thurnauer and J.T. Beatty (ed.), The purple phototrophic bacteria. Springer, New York.. 5.. Navarro Llorens, J.M., A. Tormo and E. Martinez-Garcia. 2010. Stationary phase in gram-negative bacteria. FEMS Microbiol. Rev. 34:476-495.. 6.. Oda, Y., S.-J. Slagman, W.G. Meijer, L.J. Forney and J.C. Gottschal. 2000. Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris. FEMS Microbial. Ecol. 32:205-213.. 7.. Weber, H., T. Polen, J. Heuveling, V.F. Wendisch and R. Hengge. 2005. Genome-wide analysis of the general stress response network in Escherichia coli: σS-dependent genes, promoters, and sigma factor selectivity. J. Bacteriol. 187:1591-1603.. 4.

(11) CHAPTER I. Light-dependent survivability in purple photosynthetic bacteria under carbon starvation conditions. 5.

(12) SUMMARY. Survivability and ATP levels under carbon-starvation conditions were investigated in purple non-sulfur photosynthetic bacteria, Rhodopseudomonas palustris CGA009 and Rhodospirillum rubrum S1T. Both species of purple bacteria survived longer in the light when compared to the survival in the dark. ATP levels in the cultures were maintained in the light and clearly down shifted when illumination was shut down. In the dark, a parallel decrease of CFU and the ATP level was observed in both species, although more days were needed for the decrease in R. palustris CGA009 compared to in R. rubrum S1T. These observations indicate that the bacterial cells survive effectively in natural environments when ATP is produced by photosynthesis even if carbon sources are depleted.. 6.

(13) INTRODUCTION. Purple non-sulfur photosynthetic bacteria belong to the alpha and beta subgroups of Proteobacteria. Their energy metabolisms are characterized by anoxygenic photosynthesis and they utilize organic carbon compounds as a carbon source for growth. Purple bacteria also have the ability to obtain energy through fermentation, aerobic respiration, and anaerobic respiration (e.g., denitrification) (14). They are widely distributed in natural environments (fresh and marine waters, lagoons, sediments, soil and so on) and are ecologically important since they substantially contribute to carbon, nitrogen, and sulfur cycles on the earth (16). Their ecophysiology is a subject of considerable interest. Responses of metabolisms of the purple bacteria to environmental changes have been investigated (1, 7). However, susceptibility of the bacteria to environmental stresses has been poorly characterized. Among potential stresses, the response to starvation is very important for microbes in natural environments, as their environments are frequently depleted of nutrients (12) and bacteria often experience a sudden nutrient depletion (e.g., carbon starvation) in the actively growing state. Some reports have discussed the survivability of purple bacteria under starvation conditions (4, 17). Breznak et al. reported that carbon-starved cells of Rhodospirillum rubrum strain Ha maintained viability longer in the light than that in the dark (4). Oda et al. observed the positive effect of illumination on the survivability of another species of purple bacteria, Rhodopseudomonas palustris strain DCP3 (17). Purple non-sulfur anoxygenic 7.

(14) phototrophic bacteria use a photosystem II type reaction center complex for photosynthesis, and it is expected that they can produce ATP by cyclic photophosphorylation without organic nutrients under illumination (Fig. I-1). Positive effect of light on starvation survival have been reported in some aerobic anoxygenic phototrophs, which are heterotrophs and carry out anoxygenic photosynthesis under oxic conditions but never as their energy source for growth (9, 13, 19, 20). Proteorhodopsin-containing bacteria are also heterotrophs and use light as an additional energy source and some species seemed to use light energy for survival under starvation conditions (6, 10). Although energy supply by photosynthesis under illumination seemed to promote survivability in various bacterial groups, the relationship between energy level and survivability remains obscure. In this study, I determined the viability and ATP levels of purple non-sulfur photosynthetic bacteria in a non-growing state under carbon-starvation conditions in the light and dark. To clarify the effect of sudden nutrient depletion on the viability and ATP levels of the starved cells, the starved cultures were prepared as follow; (i) the purple bacteria were grown in a culture medium containing a low concentration of the carbon source in the light, (ii) when the increase in optical density ceased after the exponential growth phase due to the depletion of the carbon source, the culture was defined as being in carbon-starvation conditions.. 8.

(15) Fig. I-1. Simplified scheme of electron flow in anoxygenic photosynthesis in a purple photosynthetic bacteria. Light energy converts a weak electron donor, P870, into a very strong electron donor, P870 . Bchl, Bacteriochlorophyll; Bph, Bacteripheophytin; QA, QB, intermediate quinones; Q pool, quinone pool in membrane; Cyt, cytochrome. The result of electron transport is thus the generations of a proton motive force that driving ATP synthesis.. 9.

(16) MATERIALS AND METHODS. Bacterial strains and preparation of starved cells. Rhodopseudomonas palustris strain CGA009 (= ATCC BAA-98) and Rhodospirillum rubrum strain S1T (= ATCC11170) were used in this study. A carbon-limited medium (pH 7.0) containing (per liter) 0.5 g sodium succinate as the sole source of carbon, 1 g (NH4)2SO4, 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (11) and 5 mL of a basal salt solution (11) was used to prepare carbon starved cells. Purple bacteria used in this study were cultivated in 150-mL glass vials containing 120 mL of carbon-limited medium and maintained at 30°C using a waterbath under illumination [tungsten lamp with 750 nm longpass filter; 600 J s-1 m-2, quantitated by pyranometer (LI-190SA, Meiwafosis, Tokyo, Japan)]. The vials were sealed with butyl rubber stoppers and aluminum seals after replacing the gas phase with N2 gas. The culture solution was continuously agitated using a magnetic stirrer. Bacterial growth was monitored by determining optical density at 660 nm. When the increase in optical density ceased after the exponential growth phase due to depletion of the carbon source (i.e., succinate), the culture was defined as being in carbon-starvation conditions. The starved cells in vials were incubated at 30°C with agitation in the light as described above or in the dark. A portion of the culture solution was collected from the vial to determine the viability and the ATP level.. 10.

(17) Colony forming units (CFUs). CFUs were determined by plate count. A diluted culture solution was spread on a 1.5% agar plate containing (per liter) 1 g sodium succinate, 1 g sodium acetate, 0.1 g yeast extract (Nihon Seiyaku, Tokyo, Japan), 0.1g Na2S2O3 5H2O, 1 g (NH4)2SO4, 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (11) and 5 mL of a basal salt solution (11). These plates were incubated aerobically at 30°C in the dark for approximately one week.. Quantification of ATP. The amount of ATP in the culture solution was quantified by means of the luciferase reaction using the BacTiter-Glo Microbial Cell Viability Assay Kit (Promega, Madison, WI, USA) and GloMax 20/20n Luminometer (Promega) or using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science, Indianapolis, IN, USA) and infinite 200 Multimode Microplate Reader (Tecan, Research Triangle Park, NC, USA).. 11.

(18) RESULTS. Survivability of R. palustris and R. rubrum under carbon starvation conditions in the light and dark. R. palustris CGA009 and R. rubrum S1T were grown photoheterotrophically (Fig. SI-1). When sufficient organic carbon is available (5 g sodium succinate per liter), the optical density increased to over 0.5 (Fig. SI-1). In R. palustris CGA009, when initial succinate concentration was reduced to 0.5 g sodium succinate per liter, a significant part of the succinate was reduced to fumarate which was observed in the growing culture. Then the growth stopped at the optical density of around 0.2-0.3 after depletion of succinate and converted fumarate (Fig. SI-1, SI-2). The cells in growth-stopped culture were defined as carbon starved cells. CFUs of the carbon-starved cells were determined after incubation in the light and dark (Fig. I-2). In both species, higher CFU values were maintained in the light compared to CFUs in the dark. CFUs on day 20 were 92.4% and 25.6% of the initial values in R. palustiris CGA009 and R. rubrum S1T, respectively (Fig. I-2). Decreasing rates of CFUs in the dark were different between both species (Fig. I-2); R. palustris CGA009 showed clearly higher survivability in the dark compared to R. rubrum S1T (Fig. I-2). R. palustris CGA009 was able to keep a constant number of CFUs under the dark conditions for 5 days, and then the CFUs gradually decreased to reach 5.4% of the initial value on day 16 and 0.05% on day 26 (Fig. I-2a). CFUs of R. rubrum S1T rapidly decreased in the dark and reached 0.02% of the initial values after 3 days of starvation 12.

(19) (Fig. I-2b). The results are similar to those in earlier studies of starvation survival in both species (4, 17).. ATP level of R. palustris and R. rubrum under starvation conditions in the light and dark. ATP levels of the cultures were also determined in R. palustris CGA009 and R. rubrum S1T (Fig. I-2). In the light, the ATP level of R. palustris CGA009 was 62% of the day 0 value on day 7 and further reduction to 46% after 20 days was observed when the CFUs were 92% of the initial value (Fig. I-2a). In the dark, although CFUs were 92% of day 0 value on day 5, ATP concentrations had decreased to 11% of the day 0 value. By day 20, both ATP and CFUs had decreased to 0.3% of their day 0 values. In R. rubrum S1T, on the other hand, the ATP level in the dark rapidly decreased to 0.3% of that on day 0 on day 6 and the CFUs were reduced to 0.007% (Fig. I-2b). Infrared absorption spectra of intact cells show absorption peaks of bacteriochlorophylls bound to the light-harvesting photopigment complexes. Figure SI-3 shows absorption spectra of R. palustris CGA009 and R. rubrum S1T cells under starvation conditions in the light and dark. In the light on day 20, the starved cells of R. palustris CGA009 showed typical two absorption peaks of bacteriochlorophyll at 806 and 860-864 nm. This indicates that photosynthetic apparatus remained as intact under the starvation conditions. Furthermore, the starved cells in the dark show similar spectra pattern to that in the light. The starved cells of R. rubrum S1T in the light and dark also showed typical absorption peaks at 880-882 nm. If light-harvesting photopigment 13.

(20) complexes had been broken, it was expected that absorption bounds of bacteriochlorophyll were jumbled and shifted. The present results indicated that light-harvesting photopigment protein complexes were maintained in the membrane and a membrane containing those protein complexes was also probably maintained even if cells lost cellular survivability.. 14.

(21) (a))R.#palustris% 9% 1.0E+09%. Light%. #10 % 1E#10%. 7% 1.0E+07%. #12 % 1E#12%. Log10CFU)mL+1. #11 % 1E#11%. Log10ATP)mL+1. 8% 1.0E+08%. Dark%. 1.0E+06% 6%. #13 % 1E#13%. 1.0E+05% 5%. #14 % 1E#14%. 1.0E+04% 4%. #15 % 1E#15%. 0%. 10%. 20%. 30%. Days (b))R.#rubrum# ) 8%. 1.0E+08. Light%. 5.0E-11 #11 %. 6%. 5.0E-12 #12 %. 1.0E+06. Dark%. 5%. 1.0E+05. 5.0E-13 #13 %. 1.0E+04. 4%. 5.0E-14 #14 %. 3%. #15 % 5.0E-15. 1.0E+03. 0%. 10%. Days. 20%. 5×Log10ATP)mL+1). 7%. 1.0E+07. Log10CFU)mL+1). 5.0E-10 #10 %. 30%. Fig. I-2. Change in ATP level (gray line) and CFU (black line) during carbon-starvation conditions; for R. palustris CGA009 (a) and R. rubrum S1T (b). Starved cells were incubated in the light (open symbol) and dark (filled symbol). Time 0 was defined as the time when the growth stopped. The amount of ATP in the culture solution was quantified by means of the luciferase reaction using the BacTiter-Glo Microbial Cell Viability Assay Kit (Promega, Madison, WI, USA).. 15.

(22) Effect of illumination or darkness on ATP level in starved cells. To estimate the ability of ATP generation by light in the starved cells, cultures were illuminated or kept in the dark for 1 minute when cells were collected from culture and then ATP levels were determined. When cells of R. palustris CGA009 maintained in the light for 5 days were shut down the illumination for 1 minute, the ATP level decreased to 64% of that of cells illuminated (Fig. I-3a). Similar reduced values were observed on day 10, 15 and 20. This may suggest that over 30% of ATP in the starved cells were synthesized and consumed rapidly under the light. On the other hands, when cells maintained in the dark for 5 days, in which CFU was still maintained, and illuminated for 1 minute, ATP levels largely increased to 10-fold (Fig. I-3b). This suggests that starved cells in the dark still had ability of photosynthesis for several days even after the dark ATP level was largely decreased. After 15 days of starvation in the dark, this up-shift of ATP level by illumination became smaller, and on day 20 this up-shift of ATP level was scarcely observed (Fig. I-3b). In R. rubrum S1T, similar changes of ATP levels were observed in the light and dark (Fig. I-4). When cells, that were maintained in the light for 5 days after the beginning of starvation, were transferred to the dark condition, the ATP level was slightly decreased and ATP level was maintained over 80% of that of cells continuously illuminated until the ATP extraction (Fig. I-4a). When starved cells maintained in the dark for 3 days were illuminated for 1 minute, ATP levels increased up to 5-fold. On day 5, the increase of this ATP level became insignificant (Fig. I-4b).. 16.

(23) ATP)(nmol/mL)%. (a)% 10.0%%. Light%. 1.0%%. 0.1%% 0%. 5%. 10%. 15%. 20%. 25%. Days%. ATP)(nmol/mL)%. (b)% 1.000%%. Dark%. 0.100%%. 0.010%%. 0.001%% 0%. 5%. 10%. 15%. Days%. 20%. 25%. Fig. I-3. Effect of illumination or darkness on ATP level of starved cells of R. palustris CGA009. (a) Starved cells were maintained in the light conditions. When cells were collected from the vial, the vial was under the light (solid line) or was shut down the illumination for 1 minute (closed diamonds) and then a portion of the culture solution was collected from the vial. (b) Starved cells were maintained in the dark conditions. When cells were collected from the bottle, the bottle was under the dark (solid line) or was illuminated for 1 minute (closed diamonds) and then a portion of the culture solution was collected from the vial. The amount of ATP in the culture solution was quantified by means of the luciferase reaction using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science, Indianapolis, IN, USA).. 17.

(24) (a)% 1.00% ATP)(nmol/mL)%. Light%. 0.10% 0%. 1%. 2%. 3%. Days%. 4%. ATP)(nmol/mL)%. (b)% 1.000%%. 5%. 6%. Dark%. 0.100%%. 0.010%%. 0.001%% 0%. 1%. 2%. 3%. Days%. 4%. 5%. 6%. Fig. I-4. Effect of illumination or darkness on ATP level of starved cells of R. rubrum S1T. (a) Starved cells were maintained in the light conditions. When cells were collected from the vial, the vial was under the light (solid line) or was shut down the illumination for 1 minute (closed diamonds) and then a portion of the culture solution was collected from the vial. (b) Starved cells were maintained in the dark conditions. When cells were collected from the bottle, the bottle was under the dark (solid line) or was illuminated for 1 minute (closed diamonds) and then a portion of the culture solution was collected from the vial. The amount of ATP in the culture solution was quantified by means of the luciferase reaction using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science, Indianapolis, IN, USA).. 18.

(25) DISCUSSION. In this study, I characterized the survivability and ATP levels under carbon-starvation conditions in purple bacteria, R. palustris CGA009 and R. rubrum S1T in the light and dark. As reported previously in R. palustris strain DCP3 (17) and R. rubrum strain Ha (4), illumination helped to maintain CFU in the starved cells of R. palustris CGA009 and R. rubrum S1T (Fig. I-3). As determined for starved cells of those species, ATP levels were maintained in the light (Fig. I-2) and ATP levels were clearly down shifted when illumination was shut down (Fig. I-3a. I-4a). In addition, photosynthetic apparatus seemed to be remained intact in cells even under starvation conditions (Fig. SI-3). This finding indicated that ATP was produced in the light without organic nutrients through cyclic photophosphorylation. It is expected that the ATP produced in the light may be consumed to keep viability by maintaining cytoplasmic homeostasis and/or synthesizing mending proteins under the non-growing conditions. In R. rubrum S1T, the ATP level was slightly decreased in the light while the CFU was decreased. The rapid decrease of ATP in the dark in R. rubrum S1T is probably the main reason for the rapid decrease of the viability after starvation. In a previous study, similar tendency of CFU and ATP level concomitant declining was observed in non-photosynthetic heterotrophic bacteria (5, 8). In this study a parallel decrease of CFU and the ATP level was also observed in R. palustris CGA009, although more days were needed for the decrease compared to in R. rubrum S1T. 19.

(26) After 5 and 10 days from the beginning of the starvation in R. palustris CGA009, CFU slightly decreased to 92% and 51% although the ATP level decreased more considerably to 11% and 7%, respectively. When cells maintained in the dark for 10 days were illuminated for 1 minute, ATP levels increased more than 10-fold. The decrease of ATP concentration in cells until a certain level, e.g. 10% of the amount under the growing conditions, may not be so fatal to survival. Decline of viability in the dark might be caused by programmed cell death; carbon-starvation and the cell density could be an inducing factor of programmed cell death. It was reported that programmed cell death were induced by production of toxic proteins (15, 18). Reason why starved cells were able to maintain viability in the light might be protecting them using ATP from programmed cell death system. It seems that relationship between energy level and bacterial survival is not so simple; it was reported that energy charge was not well related to survival all time and energy states of cells affect various bacterial physiology and the gene regulation under starvation (2, 3, 21, 22). Although the detail of the effects of light on starvation survival of purple bacteria should be investigated further, the photosynthetic bacterial group is one of the suitable bacterial groups to test the effect of the energy level on physiology of starved cells since their energy level are easily controlled by light intensity. In natural environments, bacteria are subjected to be placed frequently under nutrients depleted conditions. Oligotrophic environments are often observed in the open ocean. Aerobic anoxygenic phototrophs and proteorhodopsin-containing bacteria, which are widely distribution in ocean surface water, seem to adapt to these oligotrophic 20.

(27) environments using light energy as an additional energy (6, 9, 10, 13, 19, 20). In addition to those bacterial groups, purple non-sulfur anaerobic anoxygenic photosynthetic bacteria should be able to survive using light energy in nutrient depleted environments. It may be possible that light energy is useful energy source for bacterial starvation-survival. In summary, two species of purple bacteria showed high viability and ATP levels under long-term carbon starvation conditions in the light. This indicates that they effectively survive in natural environments where light energy is available even if carbon sources are depleted. Although it has not been clarified how ATP produced in the light affects viability, our results suggested that maintaining ATP level is critical for the starvation-survival in purple bacteria. Physiological characterization of non-growing cells under the starved conditions should be performed in more detail to understand the distribution and abundance of purple photosynthetic bacteria in changing environments.. 21.

(28) SUPPLEMENTAL MATERIALS R.#palustris!. Growth)(OD660)%. 10.00%. 1.00%. 0.10%. 0.01% 0%. 50%. 100%. 150%. Time)(h)% R.#rubrum!. Growth)(OD660)%. 1.000%%. 0.100%%. 0.010%%. 0.001%% 0%. 50%. 100%. 150%. Time)(h)% Fig. SI-1. Growth of the purple bacteria in the carbon-limited medium containing 0.5g sodium succinate per liter (solid line) or in the carbon-sufficient medium containing 5 g sodium succinate per litter (broken line). The cells were grown under anaerobic light conditions.. 22.

(29) 18.0%%. Succinate%. 500.0%%. 16.0%%. Fumarate%. 14.0%%. 400.0%%. 12.0%% 10.0%%. 300.0%%. 8.0%%. 200.0%%. 6.0%% 4.0%%. 100.0%%. Fumarate)(μmol/L)%. Succinate)(μmol/L)%. 600.0%%. 2.0%%. 0.0%%. 0.0%% 51%. 52%. 53%. 55%. 56%. 59%. Time)(h)% 0.28% 0.27% 0.26% 0.25% 0.24% 0.23% 0.22% 0.21% 0.20% 50%. 52%. 54%. 56%. 58%. 60%. Time)(h)% Fig. SI-2. Change in concentration of succinate and fumarate in culture medium of R. palustris CGA009. Cells were grown in carbon-limited medium under anaerobic light conditions. Graphs show bacterial growth from middle of the exponentially growth to growth stopping (b). After 56-hour cultivation, growth was completely stopped (b) and succinate (white bars) and fumarate (gray bars) were not detected (a). N.D., not detected. 23.

(30) Fig. SI-3. Absorption spectra of intact cells of purple bacteria under starvation conditions in the light (solid line) or in the dark (broken line). Spectrum of R. palustris CGA009 was recorded from cells on day 20 starvation (a) and that of R. rubrum S1T was recorded from cells on day 10 starvation (b). 24.

(31) REFERENCES. 1.. Arai, H., J. H. Roh, and S. Kaplan. 2008. Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4. 1. J. Bacteriol. 190:286-299.. 2.. Barrette, W. C., Jr., D. M. Hannum, W. D. Wheeler, and J. K. Hurst. 1988. Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low energy charge. J. Bacteriol. 170:3655–3659. 3.. Boes, N., K. Schreiber, E. Härtig, L. Jaensch, and M. Schobert. 2006. The Pseudomonas aeruginosa universal stress protein PA4352 is essential for surviving snaerobic energy stress. J. Bacteriol. 188: 6529–6538. 4.. Breznak, J.A., C.J. Potrikus, N. Pfennig, and J.C. Ensign. 1978. Viability and endogenous substrates used during starvation survival of Rhodospirillum rubrum. J. Bacteriol. 134:381-388.. 5.. Chapman, A.G., Fall, L. and Atkinson, D.E. 1971. Adenylate energy change in Escherichia coli during growth and starvation. J. Bacteriol.108:1072-1086.. 6.. DeLong, E.F., and Be ́ja`, O. 2010. The light-driven proton pump proteorhodopsin enhances bacterial survival during tough times. PLoS Biol. 8: e1000359.. 7.. Dubbs J.M., and F.R. Tabita. 2004. Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy generation. FEMS Microbiol. Rev. 28:353-376. 25.

(32) 8.. Eydal, H.S., and K. Pedersen. 2007. Use of an ATP assay to determine viable microbial biomass in Fennoscandian Shield groundwater from depths of 3-1000 m. J. Microbiol. Methods. 70:363-73.. 9.. Fleischman, D. E., W. R. Evans, and I. M. Miller. 1995. Bacteriochlorophyllcontaining Rhizobium species, p. 123–136. In R. E. Blankenship, M. T. Madigan, and C. E. Bauer (ed.), Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.. 10. Gomez-Consarnau, L., N. Akram, K. Lindell, A. Pedersen, R. Neutze, D.L. Milton et al. 2010. Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation. PLoS Biol. 8: e1000358. 11. Hanada, S., A. Hiraishi, K. Shimada, and K. Matsuura. 1995. Chloroflexus aggregans sp. nov., a filamentous phototrophic bacterium which forms dense cell aggregates by active gliding movement. Int. J. Syst. Bacteriol. 45:676-681. 12. Haruta, S. 2013. Rediscovery of the microbial world in microbial ecology. Microbes Environ. 28:281-284. 13. Hiraishi, A., Y. Matsuzawa, T. Kanbe, and N. Wakao. 2000. Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments. Int. J. Syst. Evol. Microbiol. 50:1539–1546. 14. Imhoff, J.F. 2006. The phototrophic alpha-proteobacteira, p. 41- 64. In M. Dworkin, S. Falkow, E. Rosenberg, K-H. Schleifer, and E. Stackebrandt (ed.), The Prokaryotes, 3rd ed., vol. 5. Springer, New York. 26.

(33) 15. Kumar, S., I. Kolodkin-Gal and H. Engelberg-Kulka. 2013. Novel quorum-sensing peptides mediating interspecies bacterial cell death. mBio. 4: e00314-13 16. Madigan, M.T., and D.O. Jung. 2009. An overview of purple bacteria: systematics, physiology, and habitats, p. 1- 15. In C. N. Hunter, F. Daldal, M. C. Thurnauer and J. T. Beatty (ed.), The purple phototrophic bacteria. Advances in photosynthesis and respiration, vol. 28. Springer, New York. 17. Oda, Y., S.-J. Slagman, W.G. Meijer, L.J. Forney, and J.C. Gottschal. 2000. Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris. FEMS Microbiol. Ecol. 32:205-213. 18. Rice, K.C. and K.W. Bayles. 2008. Molecular control of bacterial death and lysis. Microbiol. Mol. Biol. 72: 85-109. 19. Shiba, T. 1984. Utilization of light energy by the strictly aerobic bacterium Erythrobacter sp. OCh 114. J. Gen. Appl. Microbiol. 30:239–244. 20. Suyama, T., T. Shigematsu, T. Suzuki, Y. Tokiwa, T. Kanagawa, K. V. P. Nagashima, and S. Hanada. 2002. Photosynthetic Apparatus in Roseateles depolymerans 61A Is Transcriptionally Induced by Carbon Limitation. Appl. Environ. Microbiol. 68: 1665–1673 21. Wadhawan. S., S. Gautam and A. Sharma. 2010. Metabolic stress-induced programmed cell death in Xanthomonas. FEMS Microbiology Letters. 312: 176– 183.. 27.

(34) 22. Zhang, S., and W. G. Haldenwang. 2005. Contributions of ATP, GTP, and Redox State to Nutritional Stress Activation of the Bacillus subtilis σB Transcription Factor. J. Bacteriol. 187: 7554–7560. 28.

(35) CHAPTER II. Species-dependent survivability in purple photosynthetic bacteria under carbon starvation conditions. 29.

(36) SUMMARY. Survivability under carbon-starvation conditions was investigated in purple non-sulfur photosynthetic bacteria, Rhodobacter sphaeroides 2.4.1T and Rubrivivax gelatinosus. IL144,. following. the. study. of. carbon. starvation. survival. in. Rhodopseudomonas palustris CGA009 and Rhodospirillum rubrum S1T described in Chapter I. Both R. sphaeroides 2.4.1T and R. gelatinosus IL144 survived longer in the light when compared to the survival in the dark as described in the previous Chapter in R. palustris CGA009 and R. rubrum S1T. Among the four species, R. palustris CGA009, which is widely distributed in natural environments including various soils, showed higher survivability and tolerance against hypertonic stress in the dark comparing with other three species. Species dependency of the survivability during the hypertonic stress suggested that the longer survivability of R. palustris CGA009 may be related to the function of cell membrane for maintaining cytoplasmic homeostasis.. 30.

(37) INTRODUCTION. Purple non-sulfur photosynthetic bacteria belong to the alpha and beta subgroups of Proteobacteria and they are classified in 20 genera of 5 orders (17). Their energy metabolisms are characterized by anoxygenic photosynthesis and they utilize organic carbon compounds as a carbon source for growth. They are widely distributed in natural environments and are ecologically important since they substantially contribute to carbon, nitrogen, and sulfur cycles on the earth (17). In natural environments, bacterial growth is restricted due to a wide variety of environmental factors and the response to starvation is very important for microbes since their environments are frequently depleted of nutrients (9). In the previous Chapter, I have discussed the survivability of purple bacteria under starvation conditions (Fig. I-2; 3, 20). In those study, it was found that carbon-starved cells of Rhodopseudomonas palustris and Rhodospirillum rubrum maintained viability longer in the light than that in the dark. Energy supply by photosynthesis under illumination seemed to promote survivability. However, it is not clear that those phenomena are common among purple non-sulfur anoxygenic phototrophic bacteria. In this Chapter, I described the viability of two representative species of purple bacteria, Rhodobacter sphaeroides and Rubrivivax gelatinosus, in a non-growing state under carbon-starvation conditions in the light compared to in the dark. In addition, I focused decreasing-pattern of the viability in the starved cells under the dark and susceptibility against osmotic or heat stresses of the starved cells of four representative 31.

(38) species of purple bacteria was also investigated. The four species of purple bacteria used in this study belong to different orders; Rhodopseudomonas palustris strain CGA009 (the order Rhizobiales, Alphaproteobacteria), Rhodospirillum rubrum strain S1T (the order Rhodospirillales, Alphaproteobacteria), Rhodobacter sphaeroides strain 2.4.1T (the order Rhodobacterales, Alphaproteobacteria), and Rubrivivax gelatinosus strain IL144 (the order Burkholderiales, Betaproteobacteria) (Fig. II-1). These four species are mesophilic and their distributions in natural environments are different from each other (12-15).. 32.

(39) Fig. II-1. Phylogenetic distribution of purple non-sulfur photosynthetic bacteria in Proteobacteria. The phylogenetic tree was constructed based on a comparison of the nucleotide sequences of 16S rRNA genes. Purple photosynthetic bacteria are orange colored and species used in this study are boxed by blue line.. 33.

(40) MATERIALS AND METHODS. Bacterial strains and preparation of starved cells. Rhodopseudomonas. palustris. strain. CGA009. (=. ATCC. BAA-98),. Rhodospirillum rubrum strain S1T (= ATCC11170), Rhodobacter sphaeroides strain 2.4.1T (= ATCC 17023), and Rubrivivax gelatinosus strain IL144 (= NBRC 100245) were used in this study. A carbon-limited medium (pH 7.0) containing (per liter) 0.5 g sodium succinate as the sole source of carbon, 1 g (NH4)2SO4, 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (7) and 5 mL of a basal salt solution (7) was used to prepare carbon starved cells. Purple bacteria used in this study were cultivated in 150-mL glass vials containing 120 mL of carbon-limited medium and maintained at 30°C using a waterbath under illumination [tungsten lamp with 750 nm longpass filter; 600 J s-1 m-2, quantitated by pyranometer (LI-190SA, Meiwafosis, Tokyo, Japan)]. The vials were sealed with butyl rubber stoppers and aluminum seals after replacing the gas phase with N2 gas. The culture solution was continuously agitated using a magnetic stirrer. Bacterial growth was monitored by determining optical density at 660 nm. When the increase in optical density ceased after the exponential growth phase due to depletion of the carbon source (i.e., succinate), the culture was defined as being in carbon-starvation conditions. The starved cells in vials were incubated at 30°C with agitation in the light as described above or in the dark. A portion of the culture solution was collected from the vial to determine the viability and the ATP level. 34.

(41) Colony forming units (CFUs). CFUs were determined by plate count. A diluted culture solution was spread on a 1.5% agar plate containing (per liter) 1 g sodium succinate, 1 g sodium acetate, 0.1 g yeast extract (Nihon Seiyaku, Tokyo, Japan), 0.1g Na2S2O3 5H2O, 1 g (NH4)2SO4, 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (7) and 5 mL of a basal salt solution (7). These plates were incubated aerobically at 30°C in the dark for approximately one week.. Osmotic and heat treatments. In order to determine susceptibility to osmotic stress, the starved cells obtained as described above were suspended in a buffer solution containing (per liter) 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (7) and 5 mL of a basal salt solution (7), and either 2.0 M sucrose or 2.5 M NaCl. After incubation at 30°C for 10 min in the dark, CFUs were determined as mentioned above. In order to determine susceptibility to heat, CFUs were determined after incubation of the starved cells at 50°C for 30 min in the dark.. 35.

(42) RESULTS Survivability of four species of purple bacteria under carbon starvation conditions in the light and dark. R.. sphaeroides. 2.4.1T. and. R.. gelatinosus. IL144. were. grown. photoheterotrophically (Fig. SII-1). In cultures containing 0.5 g sodium succinate per liter, cells were grown exponentially until succinate deprivation caused cessation of growth; we defined these cells as carbon starved cells. CFUs of the carbon-starved cells were determined after incubation in the light and dark (Fig. II-2). In both strains, higher CFU values were maintained in the light compared to CFUs in the dark. Decreasing rates of CFUs in the dark were different between both strains (Fig. II-2); R. sphaeroides 2.4.1T showed clearly higher survivability in the dark compared to R. gelatinosus IL144 (Fig. II-2). CFUs of R. sphaeroides 2.4.1T maintained more than 70% until 3 days of starvation, decreased to 0.1% on day 15, and then to 0.005% on day 25 in the dark (Fig. II-2a). CFU of R. gelatinosus IL144 rapidly decreased in the dark and reached 0.003% of the initial value after 3 days of starvation (Fig. II-2b). R. sphaeroides 2.4.1T maintained considerably higher CFU comparing with R. gelatinosus IL144 in the dark, while R. palustris CGA009 was able to survive longer when compared to R. sphaeoides 2.4.1T (Fig. I-2a, II-2). The decreasing-pattern of survivability of R. gelatinosus IL144 in the dark was seemed similar to that of R. rubrum S1T in the dark (Fig. I-2b, II-2b). Differences in the survivability between the two species were somewhat observed even in the light, i.e., CFUs on day 20 were 37.8% and 5.4% of the initial values in R. sphaeroides 2.4.1T and R. gelatinosus IL144, respectively (Fig. II-2). 36.

(43) (a)$R.#sphaeroides" 10". Log10CFU$mL21". Light" 8". 6". Dark". 4" 0". 10". Days". 20". 30". (b)$R.#gelatinosus" 10. Log10CFU$mL21". 1.0E+10" ". Light" 8. 1.0E+08" ". 6. 1.0E+06" ". Dark" 4. 1.0E+04" ". 0". 10". Days". 20". 30". Fig. II-2. Change in CFUs during carbon-starvation conditions. Starved cells of R. sphaeroides 2.4.1T (a) and R. gelatinosus IL144 (b) were incubated in the light (open symbol) and dark (filled symbol). Time 0 was defined as the time when the growth completely stopped. Each experiment was performed in duplicate and representative values are represented.. 37.

(44) Effect of osmotic and heat stress on viability. Susceptibility to osmotic and heat stresses were compared among the four species of purple bacteria, R. palustris CGA009, R. rubrum S1T, R. sphaeroides 2.4.1T and R. gelatinosus IL144. Starved cells were prepared as described above. The cells were subjected to stress conditions in the dark and CFUs were determined (Fig. II-3, II-4). At first, to determine the effect of incubation time in osmotic-stress solution and osmotic concentration on viability of 4 species, the starved cells were incubated in 0.7 M or 2.0 M sucrose for up to 120 min (Fig. II-3). After incubation in 0.7 M sucrose, R. palustris CGA009, R. sphaeroides 2.4.1T and R. rubrum S1T maintained high viability after 10 min and then R. palustris CGA009 and R. sphaeroides 2.4.1T maintained viability more than 70% after 60 min, in contrast, viability of R. rubrum S1T gradually decreased with time and reached to less than 50% after 60 min. It has been known that when bacteria were exposed to osmotic stress, they began to synthesize and accumulate compatible solutes and change the membrane composition and then adapted to stress conditions (24, 25, 26). It may be suggested that R. rubrum S1T could resist osmotic upshift by 0.7 M sucrose but they could not adapt to the same osmotic stress conditions. In 2.0 M sucrose, R. palustris CGA009 and R. sphaeroides 2.4.1T also maintained viability more than 70% even when they were incubated for 120 min under the conditions. In contrast, viability of R. gelatinosus IL144 rapidly decreased; the viability was decreased to 8% after 10 min. R. gelatinosus IL144 were not able to resist osmotic upshift. In 2.5 M NaCl, although R. palustris CGA009 were able to survive 38.

(45) considerably higher than other three species after 10 min, viability gradually reduced depending on incubation time (Fig. II-3c and 3d). These results suggested that R. palustris CGA009 could resistant to osmotic upshift by 2.5 M NaCl for the short term but after that they were not able to adapt to the same osmotic stress conditions. In contrast, other three species could not resist the osmotic upshift. To determine the response to osmotic stress in short-term, viability of four species in high concentration of NaCl and sucrose after 10 min of treatments were compared. Fig. II-4a and b summarize the response to osmotic stress for 10 min in the four species. After 10 min incubation in 2.0 M sucrose solution, R. palustris CGA009 and R. sphaeroides 2.4.1T maintained high viability (Fig. II-4a). In contrast, the sucrose stress markedly decreased the viability of R. rubrum S1T and R. gelatinosus IL144 to 0.6% and 3.7% of the values without the stress, respectively. The osmotic stress by 2.5 M NaCl did not largely affect the viability of R. palustris CGA009 (Fig. II-4b). However, R. rubrum S1T, R. sphaeroides 2.4.1T and R. gelationosus IL144 were susceptible to 2.5 M NaCl stress for 10 min and their viability decreased to 8.2%, 13% and 5.6% of the values without the stress, respectively. To estimate the effect of incubation time in heat stress, the starved cells of R. palustris CGA009, R. sphaeroides 2.4.1T and R. gelatinosus IL144 were heated at 50°C for 10 and 120 min (Fig. II-3e). R. palustris CGA009, R. sphaeroides 2.4.1T and R. gelatinosus IL144 maintained high survivability for 10 min. Although CFUs of R. sphaeroides 2.4.1T and R. gelatinosus IL144 were decreased to none at 50°C for 120 min, R. palustris CGA009 were able to maintain 80% of its viability after 120 min. Fig. 39.

(46) II-4c shows viability of four species at 50°C heat stress for 30 min. R. palustris CGA009 also showed high resistance against the heat treatment in comparison of other three species. After heating at 50°C for 30 min, 82.4% of the CFUs of R. palustris CGA009 of the value without heat treatment were maintained. On the other hand, the other three strains were not able to survive after the heat treatment.. 40.

(47) (a)$ 120 "1.2""". 0.7$M$sucrose". %$survival". 100 1.0""" R.#palustris". 80" 0.8"". R.#sphaeroides". 60" 0.6"". R.#rubrum". 40" 0.4"" 20" 0.2"" 0" 0.0"" 10min". 30min". (b)$ 120 "1.2""". 2.0$M$sucrose". 100 1.0""". %$survival". 60min". 80" 0.8"". R.#palustris#. 60" 0.6"". R.#sphaeroides#. 40" 0.4"". R.#gela3nosus#. 20" 0.2"" 0" 0.0"" 10min". 120min". Fig. II-3. Effect of the osmotic pressure and heat stress on viability of starved cells. CFUs were determined for starved cells after exposure to 0.7 M sucrose for 10, 30, 60 min (a) and 2.0 M sucrose for 10, 120 min (b), and 2.5 M NaCl for 10, 30, 60 min (c) or 10, 120 min (d) or 50°C for 10, 120 min (e) in the dark. The results are expressed as percent of the CFUs determined after incubation for each time without exposure to the stresses.. 41.

(48) (c)$ "140". 2.5M$NaCl. %$survival". 120" 100". R.#palustris" R.#sphaeroides". 80". R.#rubrum". 60" 40" 20" 0" 10min". 30min". " (d)$120 ". 2.5M$NaCl. 100". %$survival". 60min". R.#sphaeroides". 80". R.#gelatinosus". 60" 40" 20" 0" 10min". 120min". (e)$ "120". 50. %$survival". 100". R.#palustris". 80". R.#sphaeroides". 60". R.#gelatinosus". 40" 20" 0" 10min". 120min". Fig. II-3. Continued. 42.

(49) (a)$. " 120". 2.0$M$sucrose$ ". %$survival". 100". 40" 20" 0". 2.5$M$NaCl". 120" 100" 80" 60" 40" 20" 0". " 100". %$survival". (c)$. 60". " 140". %$survival". (b)$. 80". 50$°C". 80" 60" 40" 20". n.d." n.d.". n.d.". 0". Fig. II-4. Effect of the osmotic pressure in short-term and 30 min of heat stress on viability of starved cells. CFUs were determined for starved cells after exposure to 2.0 M sucrose for 10min (a), and 2.5 M NaCl for 10 min (b) or 50°C for 30 min (c) in the dark. The results are expressed as percent of the CFUs determined after incubation for 10 min (a, b) or for 30min (c) without exposure to the stresses. The results of R. palustris CGA009, R. sphaeroides 2.4.1T and R. gelatinosus IL144 in 2.0 M sucrose and that of four species in 2.5 M NaCl are from Fig. II-3.. 43.

(50) DISCUSSION. In the study described in this Chapter, I characterized the carbon-starvation responses in two species of purple bacteria, R. sphaeroides 2.4.1T and R. gelatinosus IL144 in the light and dark. As observed for R. palustris CGA009 and R. rubrum S1T in Chapter I, illumination helped to maintain CFU in starved cells of R. sphaeroides 2.4.1T and R. gelatinosus (3, 20; Fig. I-2, II-2). These four species of purple bacteria belong to different order in Proteobacteria and have been used extensively in various microbiological and biochemical studies. The results shown in this Chapter suggested that prolongation of viability by illumination under starvation conditions is common in various purple bacteria. In the previous Chapter, it was shown that ATP levels of the starved cells of R. palustris CGA009 and R. rubrum S1T were maintained in the light (Fig. I-2). This suggested that ATP is produced in the starved cells of R. sphaeroides 2.4.1T and R. gelatinosus IL144 in the light without organic nutrients through cyclic photophosphorylation. Decreasing-pattern of survivability in the dark was clearly different among four species. In some bacteria, it was reported that starvation-survival was supported by cellular storage compounds such as polyhydroxybutyrate and glycogen (22, 23). Although some purple bacteria have been known to accumulate these storage compounds (2, 5, 16, 18), they were unlikely produced under my experimental conditions, i.e., exponential growth under nitrogen-rich and carbon limiting conditions. Further the purple bacteria used in this study have not been reported to form endospores 44.

(51) or cysts, thus such resistant structure are not expected to explain the viability of these bacteria. Difference in survivability among the four purple bacterial species is possibly related to their ability to maintain the cytoplasmic homeostasis. The difference of the survivability in the dark may be related to the response to osmotic stress. As shown in Fig. II-4, resistance to the osmotic stresses was somewhat parallel to the survivability in the dark without stresses other than carbon depletion among the four species. These observations suggested that cell membranes of R. palustris CGA009 and R. sphaeroides 2.4.1T were comparatively less permeable and able to maintain cytoplasmic homeostasis against hypertonic stress with a lower consumption of energy. In contrast, R. rubrum S1T and R. gelatinosus IL144 were highly sensitive to osmotic stresses tested in this study showing that their cell membranes were more permeable. Recently, it was suggested that changes in membrane fatty acids of some bacterial species affect tolerance against environmental stresses (1, 25). ATP could also be utilized for reconstruction of cell membranes. R. palustris CGA009 was tolerant to the heat treatment of 50°C for 120 min (Fig. II-3e). In R. palustris, some strains were reported to be thermotolerant and they were able to grow at temperature of up to around 43°C (10). Among those thermotolerant strains, R. palustris ATCC17001 is a closely related strain to R. palustris CGA009 used in this study (19). Thus it may be possible that R. palustris CGA009 is also a thermotolerant. Osmotic stress by NaCl also did not largely affect the viability of R. palustris 45.

(52) CGA009, although the other three species were susceptible (Fig. II-4). The outstanding survivability of R. palustris CGA009 under various stress conditions, including carbon starvation in the dark, may be related to the cytoplasmic membranes that can maintain cytoplasmic homeostasis with a small amount of energy. R. palustris is a commonly observed species of purple bacteria in natural environments and has been detected as a major bacterial species in various environments such as paddy soil, freshwater marsh sediments and aquatic sediments (6, 8, 21). Rhizobiales to which R. palustris belongs is a large group of soil bacteria. Some bacterial species in Rhizobiales have been known to show high survivability under purified water conditions (4, 11). Soil as a habitat for bacteria is a heterogeneous and unstable environments and nutrients and light are sparsely distributed in the micro-environments in soil. High survivability of R. palustris CGA009 as shown here enables it to outcompete other bacteria in soil. On the other hands, the major habitats of the other three purple bacteria used in this study are stagnant freshwater environments exposed to the light. For example, R. geletinosus is frequently found in stable and nutrient-rich environments such as sewage ditches and activated sludge. In summary, all the four purple bacterial species used in this study showed long starvation-survival in the light. This indicates that the survival-strategy utilizing energy produced by light is common in purple non-sulfur photosynthetic bacteria. Although it has not been clarified how ATP produced in the light affects viability, my results on the stress response suggested that the purple bacteria utilize ATP to maintain cytoplasmic homeostasis possibly through the function of cell membranes. In the dark, 46.

(53) survivability was clearly different among four species and R. palustris showed extremely high survivability. Investigating the detail of physiology in this successful survivor during starvation conditions would lead to clarify a part of the bacterial survival mechanism.. 47.

(54) SUPPLEMENTAL MATERIALS R.#sphaeroides#. Growth$(OD660)". 10.000"" 1.000"" 0.100"" 0.010"" 0.001"" 0". 50". 100". 150". Time$(h)" R.#gelatinosus#. Growth$(OD660)". 1.0000" 0.1000" 0.0100" 0.0010" 0.0001" 0". 50". 100". 150". Time$(h)" Fig. SII-1. Growth of the purple bacteria in the carbon-limited medium containing 0.5g sodium succinate per liter (solid line) or in the carbon-sufficient medium containing 5 g sodium succinate per litter (broken line). The cells were grown under anaerobic light conditions.. 48.

(55) REFERENCES. 1.. Alvarez-Ordonez, A., A. Fernandez, M. Lopez, R. Arenas, and A. Bernardo. 2008. Modifications in membrane fatty acid composition of Salmonella Typhimurium in response to growth conditions and their effect on heat resistance. Int. J. Food Microbiol. 123:212-219. 2.. Brandl, H., R.A. Gross, R.W. Lenz, R. Lloyd, and R.C. Fuller. 1991. The accumulation of poly (3-hydroxyalkanoates) in Rhodobacter sphaeroides. Arch. Microbiol. 155:337-340.. 3.. Breznak, J.A., C.J. Potrikus, N. Pfennig, and J.C. Ensign. 1978. Viability and endogenous substrates used during starvation survival of Rhodospirillum rubrum. J. Bacteriol. 134:381-388.. 4.. Crist, D.K., R.E. Wyza, K.K. Mills, W.D. Bauer, and W.R. Evans. 1984. Preservation of Rhizobium viability and symbiotic infectivity by suspension in water. Appl. Environ. Microbiol. 47:895-900.. 5.. De Philippis, R., A. Ena, M. Guastini, C. Sili, and M. Vincenzini. 1992. Factors affecting poly-beta-hydroxybutyrate accumulation in cyanobacteria and in purple nonsulfur bacteria. FEMS Microbiol. Lett. 103:187-194.. 6.. Feng, Y., X. Lin, Y. Yu, and J. Zhu. 2011. Elevated ground-level O3 changes the diversity of anoxygenic purple phototrophic bacteria in paddy field. Microb. Ecol. 62:789-799. 7.. Hanada, S., A. Hiraishi, K. Shimada, and K. Matsuura. 1995. Chloroflexus 49.

(56) aggregans sp. nov., a filamentous phototrophic bacterium which forms dense cell aggregates by active gliding movement. Int. J. Syst. Bacteriol. 45:676-681. 8.. Harada, N., S. Otsuka, M. Nishiyama, and S. Matsumoto. 2003. Characteristics of phototrophic purple bacteria isolated from a Japanese paddy soil. Soil Sci. Plant Nutr. 49:521-526.. 9.. Haruta, S. 2013. Rediscovery of the microbial world in microbial ecology. Microbes Environ. 28:281-284.. 10. Hisada,T., K. Okamura and A. Hiraishi. 2007. Isolation and characterization of phototrophic purple nonsulfur bacteria from Chloroflexus and Cyanobacterial Mats in Hot Springs. Microbes Environ. 22:405–411. 11. Iacobellis, N.S., and J.E. Devay. 1986. Long-term storage of plant-pathogenic bacteria in sterile distilled water. Appl. Environ. Microbiol. 52:388-389. 12. Imhoff, J.F. 2005. Genus I, Rhospirillum Pfennig, and Trüper 1971, 17AL. p. 1-6. In D.R. Boone, N.R. Krieg, J.T. Staley, and G.M. Garity (ed.), Bergey’s Manual of Systematic Bacteriology, 2nd ed., vol. 2. Springer, New York. 13. Imhoff, J.F. 2005. Genus I, Rhodobacter Imhoff, Trüper, and Pfennig 1984, 342VP. p. 161-167. In D.R. Boone, N.R. Krieg, J.T. Staley, and G.M. Garity (ed.), Bergey’s Manual of Systematic Bacteriology, 2nd ed., vol. 2. Springer, New York. 14. Imhoff, J.F. 2005. Genus IX, Rhodopseudomonas Czurda, and Maresch 1937, 119AL. p. 473-476. In D.R. Boone, N.R. Krieg, J.T. Staley, and G.M. Garity (ed.), Bergey’s Manual of Systematic Bacteriology, 2nd ed., vol. 2. Springer, New York.. 50.

(57) 15. Imhoff, J.F. 2005. Genus incertae sedis XV, Rubrivivax Willems, Gillis, and De Ley 1991b, 70VP. p. 749-750. In D.R. Boone, N.R. Krieg, J.T. Staley, and G.M. Garity (ed.), Bergey’s Manual of Systematic Bacteriology, 2nd ed., vol. 2. Springer, New York. 16. Liebergesell, M., E. Hustede, A. Timm, A. Steinbüchel, R.C. Fuller, R.W. Lenz, and H.G. Schlegel. 1991. Formation of poly (3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria. Arch. Microbiol. 155:415-421. 17. Madigan, M.T., and D.O. Jung. 2009. An overview of purple bacteria: systematics, physiology, and habitats, p. 1- 15. In C. N. Hunter, F. Daldal, M. C. Thurnauer and J. T. Beatty (ed.), The purple phototrophic bacteria. Advances in photosynthesis and respiration, vol. 28. Springer, New York. 18. Mukhopadhyay,. M.,. A.. Patra,. and. A.K.. Paul.. 2005.. Production. of. poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate ) by Rhodopseudomonas palustris SP5212. World J. Microb. Biot. 21:765-769. 19. Okamura, K., K. Takata and A. Hiraishi. 2009. Intrageneric relationships of members of the genus Rhodopseudomonas. J. Gen. Appl. Microbiol. 55:469-478 20. Oda, Y., S.-J. Slagman, W.G. Meijer, L.J. Forney, and J.C. Gottschal. 2000. Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris. FEMS Microbiol. Ecol. 32:205-213. 21. Oda, Y., W. Wanders, L.A. Huisman, W.G. Meijer, J.C. Gottschal and L.J. Forney. 2002. Genotypic and phenotypic diversity within species of purple nonsulfur bacteria isolated from aquatic sediments. Appl. Environ. Microbiol. 68:3467-3477. 51.

(58) 22. Povolo, S., and S. Casella. 2004. Poly-3-hydroxybutyrate has an important role for the survival of Rhizobium tropici under starvation. Ann. Microbiol. 54: 307–316. 23. Ratcliff, W.C., S.V. Kadam, and R.F. Denison. 2008. Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia. FEMS Microbiol. Ecol. 65:391-399. 24. Tsuzuki, M., O. V. Moskvin, M. Kuribayashi, K. Sato, S. Retamal, M. Abo, J. Zeilstra-Ryalls, and M. Gomelsky. 2011. Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides. Appl. Environ. Microbiol. 77: 7551–7559. 25. Tymczyszyn, E. E., A. Go ́mez-Zavaglia, and E. A. Disalvo. 2005. Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic pressure of Lactobacillus bulgaricus. Arch. Biochem. Biophys. 443:66-73. 26. Wood, J.M. 2011. Osmotic stress, p. 133-156. In G. Storz and R. Hengge (ed.), Bacterial stress responses, 2nd ed. American Society for Microbiology Press, Washington, DC.. 52.

(59) CHAPTER III. Effect of light on metabolomic and transcriptomic profile of starved cells in a purple photosynthetic bacterium Rhodopseudomonas palustris. 53.

(60) SUMMARY. In carbon starved cells of anoxygenic anaerobic bacteria, Rhodopseudomonas palustris, days of survival became longer largely by illumination which works as energy source. To examine the metabolic states in both illuminated and un-illuminated cells, 5-day starved cells, which still survived mostly under both conditions, were used. Metabolites of the central and related metabolic pathways and the transcriptional profile of starved R. palustris CGA009 were analyzed and compared between cells under the light and dark. I found that metabolic profile of starved R. palustris CGA009 cells was largely different between cells incubated with light or not. In the light, various amino acids were highly accumulated, while metabolites involved in the glycolytic pathway and the TCA cycle were in the low level. It was also observed in the light that many genes related to protein turnover were highly expressed. On the other hand, expression of inorganic-ion transporters was remarkable in the dark cells. These results suggested that active turnover of macromolecules proceeded in the starved R. palustris CGA009 cells and they were supported by light energy. The metabolites related to carbon metabolism seemed to be utilized for the biosynthesis in the light. Even in the dark cells, significant levels of macromolecule turnover seemed to proceed, but amino acids may be deficient in the cells.. 54.

(61) INTRODUCTION. Bacteria often face environmental changes such as depletion of essential nutrients and they some times enter into a non-growing state. While some bacteria are responding to starvation by forming metabolically inactive endospores or cysts, the vast majority of bacterial species are not able to induce such cell differentiation but it has been observed that they survived under starvation conditions for long period. Although some growth-arrested phase phenomena such as changes in global gene expression, cell shape, and increase in stress resistance (22, 30, 47) have been the focus of intense research, profile of metabolites in growth-arrested cells has not been well characterized. Adjustment of the physiological states to metabolically stressful conditions has been observed in some bacteria (13, 15, 20). In those studies, it was reported that switching to another energy generation system such as that from aerobic metabolism to anaerobic metabolism and/or changing in utilization of endogenous metabolites occurred, and expression of genes related to those metabolic change were regulated (5, 11, 16, 34, 47); it was expected that growth-arrested cells should require energy supply for the survival. However, it is not clear yet whether the energy level affects on the metabolism in growth-arrested cells including nutrient-starved cells and how they use the energy for survival. Rhodopseudomonas palustris is a purple non-sulfur photosynthetic bacterium that is one of the species in Proteobacteria. R. palustris is widely distributed in natural environments, preferring soil and freshwater. Their major energy metabolism is 55.

(62) characterized by anoxygenic heterotrophic photosynthesis. They can get energy from light by cyclic photophosphorylation. The studies described in Chapter 1 suggested that energy production by photosynthesis in R. palustris CGA009 promoted the survivability under starvation conditions. It was expected that energy supply by light support metabolism of starved cells to survive even when the net growth of the cells is stopped because of the endogenous carbon deficiency. Recently, the concept of “metabolome”, the comprehensive analysis of metabolite pools, begins to attract attention. Metabolome is very powerful for understanding metabolism as a whole (26, 44, 48), because the metabolome is a direct reflection of the physiological status of a cell (17, 41). Although there are few studies that focus on metabolism under nutrient starvation conditions or growth-arrested status, metabolome analysis is seemed to be useful to understand metabolic response of cells because it is expected that nutrients limitation and energy level directly affect on their various pathways of metabolism. In this study, to understand the effect of energy supply by light on metabolism in starved cells, I analyzed metabolites of central and related metabolism pathways in the starved R. palustris CGA009 cells under the light and dark comparatively. In addition, to find the genes expressed in the starved cells in the light and dark, transcriptome was analyzed using the microarray. In previous studies, global changes in transcription depending on growth phases were reported in some bacteria (3, 6, 47). In those study, various genes including genes encoding metabolic enzymes and general-stress-response. proteins. were. expressed. in. growth-arrested. phase.. Transcriptional characterization of the non-growing R. palustris CGA009 cells in the 56.

(63) light and dark should be performed to understand the relationship between energy states and bacterial survival in more detail.. MATERIALS AND METHODS. Bacterial strains and preparation of starved cells. Rhodopseudomonas palustris strain CGA009 (= ATCC BAA-98) was used in this study. A carbon-limited medium (pH 7.0) containing (per liter) 0.5 g sodium succinate as the sole source of carbon, 1 g (NH4)2SO4, 0.38 g KH2PO4, 0.39 g K2HPO4, 1 mL of a vitamin mixture (18) and 5 mL of a basal salt solution (18) was used to prepare carbon starved cells. R. palustris CGA009 were cultivated in 150-mL glass vials containing 120 mL of carbon-limited medium and maintained at 30°C using a waterbath under illumination [tungsten lamp with 750 nm longpass filter; 600 J s-1 m-2, quantitated by pyranometer (LI-190SA, Meiwafosis, Tokyo, Japan)]. The vials were sealed with butyl rubber stoppers and aluminum seals after replacing the gas phase with N2 gas. The culture solution was continuously agitated using a magnetic stirrer. Bacterial growth was monitored by determining optical density at 660 nm. When the increase in optical density ceased after the exponential growth phase due to depletion of the carbon source (i.e., succinate), the culture was defined as being in carbon-starvation conditions. The starved cells in vials were incubated at 30°C with agitation in the light as described above or in the dark. A portion of the culture solution was collected from 57.

(64) the vial to determine the metabolic and transcriptomic characteristics.. Analysis of Metabolites by CE/MS. The vials incubated for 5 days of starvation in the light and dark were cooled to 4°C for 5 min with illumination or not. The cultures (optical density at 660 nm were around 0.3, sampling volume of culture were around 120 mL) were filtered using a 0.4 mm pore size filter. The residual cells on the filter were washed twice with 10 mL of ultrapure water. The filter having residual cells was soaked in 1.6 mL of methanol in a plastic dish. The dish was sonicated for 30 sec using a SONO CLEANER 200R (Kaijo, Tokyo, Japan). The cell suspension was treated with 1.1 mL of ultrapure water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) and left as rest for 30 sec. The cell extract was transferred to a 15 mL centrifuge tube and that was centrifuged at 2300 × g for 5 min at 4°C. The 1.6 mL of upper aqueous layer was distributed to four Amicon Ultrafree-MC ultrafilter tips (Millipore, Billerica, MA, USA) and centrifuged at 9,100 × g for 120 min at 4°C to remove proteins. The filtrate was centrifugally concentrated and re-suspended in 50 µL of ultrapure water for CE-MS analysis. CE-TOFMS analysis was performed using the Agilent CE-TOFMS system (Agilent, Palo Alto, CA, USA) as described previously (27). Cationic and anionic metabolites were analyzed in each suitable condition for determination. Each metabolite was identified and quantified based on the peak information, including m/z, migration time, and peak area using MasterHands ver.2.9.0.9 (developed at Keio University). 58.

(65) Analysis of fatty acid methyl esters by GC/MS. For the measurements of total fatty acids in the staved cells, “the cells at the time of beginning of starvation” and “the starved cells” were used. The cells at the time of beginning of starvation were collected when the stopped the exponential growth was confirmed and it took about 2 h for the confirmation after the actual stop time. The starved cells were collected after 5 days of starvation in the light and dark. Cells were obtained by centrifuged for 10 min at 6000 r.p.m at 4°C. The pellet was washed twice with distilled water. The washed pellets were frozen at -20°C, and then freeze-dried with a lyophilizer. Cellular fatty acid methyl esters were extracted and purified using a fatty acid methylation kit and a fatty acid methyl ester purification kit (Nacalai Tesque, Kyoto, Japan) following the manufacturer's instructions. The fatty acid composition was determined using a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japan) equipped with a MS detector (GCMS-QP5050, Shimadzu) equipped with polyethylene glycol capillary column (HP Innowax; 30 m × 0.25 mm; 0.25 µm film thickness, Agilent Technologies, Palo Alto, CA, USA) at 70 eV in scan mode. The temperature ramp was: injector 250°C, oven initially at 60 °C, held for 2 min, heated to 120°C (30°C min−1) and then to 250°C (10°C min−1, then held for 5 min). The fatty acids were identified by comparison of retention times and mass fragmentation patterns with standard substances (Supelco, Bellefonte, PA, USA).. 59.

(66) NAD+/NADH ratio. The intracellular NADH and NAD+ were extracted and assayed by using a fluorescent NAD/NADH detection kit (Cell Technology Inc., CA, USA). Briefly, 500 µl of the cultures was collected with illumination or not and then immediately suspended in 4.5 ml cool Phosphate buffered saline solution and harvested by centrifugation. Pellets were resuspended in 200 µl of NADH or NAD extraction buffer. Next 200 µl of the NAD/NADH lysis buffer were added to all the tubes and then extracts were obtained by two times of a freeze-thaw cycle. Intracellular NADH and NAD+ were measured by following the manufacturer's instructions. NAD+ was converted to NADH. NADH reacted with nonfluorescent detection reagent to form NAD+ and the fluorescent analog that was monitored at 550 nm excitation and 595 nm emission wavelengths by using an infinite 200 Multimode Microplate Reader (Tecan, Research Triangle Park, NC, USA).. Transcriptome analysis using DNA microarrays. (i) Printing of whole-genome DNA microarrays. The microarrays used in this study were custom-made R. palustris CGA009 microarrays using the 15K platform developed by Agilent Technologies. The custom-made R. palustris CGA009 microarrays were designed using Agilent's eArray web design application that support to design the custom microarray. A total of 14,823 spots represented 4,887 R. palustris CGA009 open reading frames, meaning that 99.8% of the predicted chromosomal and plasmid R. palustris CGA009 open reading frames (NCBI accession number 60.