Growth$(OD 660)"

SUPPLEMENTAL MATERIALS

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.

0.001""

0.010""

0.100""

1.000""

10.000""

0" 50" 100" 150"

0.0001"

0.0010"

0.0100"

0.1000"

1.0000"

0" 50" 100" 150"

REFERENCES

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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.

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Bacteriol. 134:381-388.

4. Crist, D.K., R.E. Wyza, K.K. Mills, W.D. Bauer, and W.R. Evans. 1984.

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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 O

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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

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, 17

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Systematic Bacteriology, 2nd ed., vol. 2. Springer, New York.

13. Imhoff, J.F. 2005. Genus I, Rhodobacter Imhoff, Trüper, and Pfennig 1984, 342

^VP

. 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,

119

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. 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.

15. Imhoff, J.F. 2005. Genus incertae sedis XV, Rubrivivax Willems, Gillis, and De Ley 1991b, 70

^VP

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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.

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.

CHAPTER III

Effect of light on metabolomic and transcriptomic profile of starved cells in

a purple photosynthetic bacterium Rhodopseudomonas palustris

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.

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

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

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 (NH

4

)

2

SO

4

, 0.38 g KH

2

PO

4

, 0.39 g K

2

HPO

4

, 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 N

2

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 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).

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).

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

NC_005296, NC_005297) are represented on the microarray. Three probes per an open reading frame were designed and represented on the array. Eighteen probes per 6 genes were spotted 10 times on array as control probes.

(ii) RNA isolation and precipitation. The vials containing culture were cooled to around 4°C for 5 min. The 30 mL of culture were centrifuged for 15 min at 10,000 r.p.m at 4°C and frozen at -80°C until RNA isolation. Cells were mixed with 750 µl of precooled TPM buffer [50 mM Tris- HCl (pH 7.0), 1.7% (wt/vol) polyvinylpyrrolidon K25, 20 mM MgCl

2

], and 0.1 g of zilconic-silica beads (0.1 mm diameter). The mixture was shaken for 60 s at maximum speed in a bead beater (Mini-beadbeater, Biospec products, Bartlesville, OK, USA). Zilconic-silica beads and cell debris were pelleted by centrifugation (5 min, 15,000 g, 4°C), and the supernatant was discarded since there was little amount of nucleic acid. The pellet was resuspended in 700 µl of a phenol-based lysis buffer [5 mM Tris-HCl (pH 7.0), 5 mM Na

2

EDTA;

0.1% (wt/vol) sodium dodecyl sulfate, 6% (v/v) water-saturated phenol], followed by a second round of bead-beating. After centrifugation, the supernatants of the phenol bead-beating treatments were extracted with water-saturated phenol ×2 and chloroform–

isoamyl alcohol [24:1 (v/v)]. The 600 µL of total nucleic acids were mixed with 600 µL

of ethanol and purified using the PureLink RNA mini kit (Life Technologies, Carlsbad,

CA, USA). DNase I digestion was performed (Takara, Shiga, Japan). RNA

concentration and quality were assessed using Agilent Bioanalyzer 2100 (RNA 6000

Nano LabChip kit, Agilent Bioanalyzer 2100; Agilent Technologies, CA, USA). The

total RNA was concentrated by ethanol precipitation, and vacuum dried and

resuspended to TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.5).

(iii) cDNA Synthesis and labeling, and hybridization. Cy3 labeling of the

cDNAs was performed with the FairPlay III Microarray Labeling Kit (Stratagene, La

Jolla, CA, USA ) and CyDye Cy3 mono-Resctive Dye (GE Healthcare,

Buckinghamshire, UK ) according to the manufacturer's instructions. Final cDNA

yields and the Cy3 incorporation rates were determined by Nanodrop analysis (Isogen

Life Science, IJsselstein, The Netherlands). Custom-made R. palustris microarrays were

hybridized with labeled cDNA. After hybridization at 60°C for 17 h, the microarrays

were washed according to the manufacturer's instructions. Then slides were scanned

using an Agilent microarray scanner, and data were extracted from the scanned

microarrays with Feature Extraction software.

ドキュメント内 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 (ページ 54-68)