(b) Abundance Abundance - The objective of this study is to clarify the relationship between su

Fig. III-3. Representative GC-MS chromatograms of medium to long chain fatty acids of starved R. palustris CGA009 cells in the light (a) and dark (b) for 5 days starvation.

Identified fatty acids were: hexadecanoic (palmitic) acid (C16:0), hexadecenoic (palmitoleic) acid (C16:1), octadecanoic (stearic) acid (C18:0) and octadecenoic (oleic or vaccenic) acids (C18:1). Retention time of 16.13 min (arrow) may be cyclic fatty acids such as cyclopropyl fatty acids but it was not identified in this study.

(a)

(b)

Table III-3. Change in ratio of unsaturated fatty acids in R. palustris CGA009 cells

under carbon starvation conditions.

Fatty acids concentration (µg mL

^-1

)

Fatty acids Beginning

of starvation

Dark 5 day Light 5 day

C16:0 0.44 0.48 0.70

C16:1 0.12 0.11 0.09

C18:0 0.46 0.49 0.65

C18:1 4.40 4.20 3.64

Total concentration 5.41 5.28 5.08

Unsaturated degree %

83.5 81.7 73.5

Metabolites were extracted from starved culture maintained in the light or dark conditions for 5days.

“Beginning of starvation” was defined as the time when the growth completely stopped.

Unsaturation degree % means the ratio of sum of C16:1 and C18:1 to total fatty acids.

Effect of light on RNA transcription of starved cells.

To understand the transcriptional activity of cells under starvation conditions, total RNA was extracted from the growing cells and the starved cells of R. palustris CGA009 (Table III-4). Total amount of RNA in growing cells was 4.96 µg mL

^-1

OD

660-1

. Total amount of RNA in the starved cells was low compared to the growing cells; after 5 days of starvation, the amount of total RNA extracted from the starved cells in the light and dark were 1.95 and 1.09 µg mL

^-1

OD

660-1

, respectively. In previous studies, a low level of RNA was observed in the growth arrested bacteria compared to the exponentially growing cells (32, 45). In addition, it is known that the degradation of rRNA is associated with starvation (10). Thus the decrease of proportion of rRNA may mainly contribute the decrease of the total RNA in starved R. palustris CGA009.

The proportion of rRNA, mRNA and tRNA was estimated in this study using

the amount of total RNA and the values of rRNA, mRNA and tRNA from the

microarray analysis. In the starved cells in the light, mRNA account for 40% of total

RNA, while the starved cells in the dark had a low level of mRNA (23.9%). Since the

starved cells in the dark have some amount of mRNA even if the lower level, it seems

likely that they are active and proteins are being synthesized even when energy supply

by light is absent.

Table III-4. RNA levels of starved cells and growing cells in R. palustris CGA009.

Total RNA were extracted from the starved cells and the growing cells, as described at Materials and Methods.

Concentrations of rRNA, mRNA, tRNA are calculated using results of microarray analysis. rRNA; rpa_RNA50-52, 57, 58. tRNA; rpa_RNA1-43, 46-49.

Concentration of RNA (µg OD

660-1

mL

^-1

)

Conditions Total RNA

rRNA

mRNA

tRNA

Dark 5day 1.09 0.80 0.26 0.02

Light 5day 1.95 1.03 0.78 0.14

Exponential

growth phase 4.96 - - -

Microarray analysis; different transcriptomic pattern of the starved cells in the light and dark.

To understand what genes are expressed in the starved cells in which metabolic profile was drastically different by illumination, transcriptome was analyzed using microarray. Total RNA was extracted from samples as described above. As described in Materials and Methods, cDNA was synthesized, labeled and hybridized to the microarray slide customized for R. palustris CGA009 in this study. Fig. SIII-1 and Table SIII-1 show the quality of RNA used for microarray analysis. Critical degradation of RNA was not observed in electrophoresis (Fig. SIII-1). Although the incorporation rate of Cy3 into cDNA was sufficient, total amount of labeled cDNA was marked low levels compared to recommended value (Table SIII-1). Thus, I focused on the highly expressed genes in the light and dark, 638 and 646, respectively, and sum of the signal values of those genes were 90% of sum of the total signal value.

The all high expression genes were Table SIII-4. Those genes were categorized to Clusters of Orthologous Groups (COG) classifications. Fig. III-4 shows the highly expressed genes categorized in functional groups, according to the COG classifications.

Both in the light and dark, the ratio of the category of Cell Division and Chromosome Partitioning was very low that probably reflects the stop of cell division.

Each COG that was abundant in the light compared to in the dark were covered

over 5% of the total high expression genes; RNA (7.8% in the light vs 6.8% in the dark),

Transport and Metabolism of Amino Acids (7.5% vs 5.6%), Translation Ribosomal

Structure and Biogenesis (5.6% vs 3.4%), Posttranslational Modification Protein

Turnover Chaperones (6.9% vs 4%). It seemed that the results of transcriptional analysis in which the category of protein and amino acids turnover was highly expressed in the light was in line with the results of metabolome analysis in which most amino acids were more accumulated in the light (Fig. III-1). It is also noteworthy that, the category of Inorganic Ion Transport and Metabolism was remarkable in the dark compared to that in the light (Fig. III-4).

Microarray analysis; characteristics of transcribed genes of the starved cells in the light and dark.

To understand the details of transcriptional characteristics, the functions of each gene that was highly expressed in the starved cells in the light and dark were examined. Among 638 highly expressed genes in the starved cells in the light, 48 genes were belonging to COG category of Amino Acids Transport and Metabolism and included 20 genes that related to transport system (Table III-5). Among 48 genes, 13 genes were also highly expressed in the dark and 8 genes were related to transport system. Genome information is available for R. palustris CGA009, and it is noteworthy that 15% genes of the genome are relevant to membrane transport system (25).

Significant numbers of transport system related genes were expressed in the starved

cells, which may be one of the carbon starvation responses; it has been considered that

oligotrophic bacteria tried to take up extracellular carbon source by various transporters

(50). In the dark, genes for membrane translocators of inorganic ions were transcribed

(Table III-5); it was possible that un-illuminated starved cells tended to maintain

Fig. III-4. Transcriptomic profile of starved R. palustris CGA009 cells in the light and dark. Highly expressed genes in the light (638 genes) and dark (646 genes) were categorized to NCBI Clusters of Orthologous Groups (COG). Coverage (category %) for all categories of the COG scheme is indicated as the ratio of the number of detected genes in each category to the total number of highly expressed genes. Orange bars, high expressed genes in the light; blue bars, high expressed genes in the dark in each COG category.

intracellular environment using translocators of inorganic ions.

In the category of Translation, Ribosomal Structure and Biosynthesis, many genes related to heat shock proteins and chaperones were highly expressed in the light (Table III-5). Highly expressed genes related to DNA damage response as lexA and recA (rpa2903, rpa3851), DNA protection as dps (rpa1274) and protection against oxidative stress as katG and sodC (rpa0429, rpa0225) were also observed in the light (Table SIII-4, p.106, 109, 112). The development of multiple stress resistance system has been reported in growth-arrested cells in other gram-negative bacteria (6, 30). The expression of stress response genes in R. palustris CGA009 may be one of the starvation responses and expression of those genes might be enhanced by light. It is noteworthy that genes of groEL and groES, that encoded heat shock protein, were highly expressed both in the light (rpa1140, rpa2164 and rpa2165) and dark (rpa2164 and rpa2165). It was reported that GroEL and GroES were expressed in the cells of R.

palustris CGA009 in various conditions including both in the growing cells and the growth-arrested cells (43). Thus, expression of those genes might be not caused by starvation response.

Recently, it was reported that EcfG controlled the general stress response in

some of the alpha subgroup of Proteobacteria (39). In addition, it was reported that

EcfG (SigT), CtrA and FixK regulators related to gene expression under carbon

starvation conditions in an alpha subgroup of Proteobacteria, Caulobacter crescentus

(6). In the present study, ecfG-like gene (rpa4225), ctrA (rpa1632) and fixK (rpa4250)

were highly expressed in the light (Table SIII-4, p.106, 112). It may be possible that

those transcription regulators relate to the high expression of genes under the light in the starved R. palustris CGA009 cells.

Purple photosynthetic bacteria have a photosynthetic gene cluster.

Photosynthetic reaction center are corded in pufLMH and bacteriochlorophyll pigment binding proteins are corded in pucAB. In the starved cells, some genes encoding photosynthetic reaction center and bacteriochlorophyll pigment binding proteins were highly expressed both in the light and dark (Table III-5). The starved cells of R.

palustris CGA009 in the light and dark showed the absorption peaks of bacteriochlorophyll (Fig. SI-3). Those results suggested that the turnover of light-harvesting photopigment complexes occurred in the starved cells both in the light and dark.

Various enzymes involve in the redox reactions and maintaining redox homeostasis in bacteria. Maintaining the redox states is important for the growth and survival (19, 24, 28, 33, 42). In R. palustris, carbon fixation reaction and hydrogen production by nitrogenase were important to maintaining the redox homeostasis in the growing cells (28, 29), and a parts of those enzyme complexes were expressed in the light (rpa1559 and rpa4620) and dark (rpa1437) in the present study (Table III-6). R.

palustris CGA009 has five genes related to CO monodehydrogenase enzymes that are known as redox enzymes. Under the light conditions, four genes of CO monodehydrogenase (rpa4666, rpa4667, rpa3802 and rpa3803) were more highly expressed (Table III-6).

Some genes relating to acetate and ethanol biosynthesis were highly expressed

in the light (rpa2153 and rpa1205) (Table IIIS-4, p.100). Under the dark conditions, the

supernatant of culture contained a small amount of fumarate and mRNA for the

succinate dehydrogenase complex that works the redox changes from succinate to

fumarate was highly expressed. In other bacteria, it was reported that genes related to

energy metabolism were expressed even if cells in the growth arrested phase (11, 20,

37); the starved cells should require energy supply for the survival. Genes encoding the

enzymes related to denitrification were highly expressed in the dark (rpa1453, rpa1455

and rpa4145) (Table III-6). It was expected that anaerobic starved cells in the dark

required other anaerobic energy metabolism than photosynthesis since they were not

able to synthesize ATP by light.

Table III-5. The high expressed genes categorized selected COGs in starved R.

palustris CGA009 cells in the light and dark.

The high expressed genes in the starved cells of R. palustris in the light

Acc. No. definition signal %^a

Amino Acid Transport and Metabolism

rpa3033 possible acetylornitine deacetylase 1.020

rpa0870 putative ornithine decarboxylase 0.908

rpa4020^b possible branched-chain amino acid transport system permease protein 0.457 rpa3810 putative periplasmic binding protein of ABC transporter 0.163 rpa1789 putative branched-chain amino acid transport system substrate-binding protein 0.106

rpa2966 nitrogen regulatory protein P-II 0.103

rpa3093 possible urea/short-chain binding protein of ABC transporter 0.092 rpa3725^b possible leucine/isoleucine/valine-binding protein precursor 0.081

rpa2046 2-isopropylmalate synthase 0.071

rpa2446 putative aminotransferase 0.069

rpa1798^b putative periplasmic binding protein for ABC transporter for branched chain

amino acids 0.058

rpa0230 aspartate-semialdehyde dehydrogenase 0.055

rpa0668 putative ABC transporter subunit, substrate-binding component 0.055

rpa2503 possible aminotransferase 0.047

rpa4807 possible branched-chain amino acid transport system substrate-binding protein 0.042 rpa3297^b possible branched-chain amino acid transport system substrate-binding protein 0.041

rpa4331 aspartate aminotransferase A 0.040

rpa0557 cysteine synthase, cytosolic O-acetylserine(thiol)lyase 0.039 rpa1664 Glyoxalase/Bleomycin resistance protein/dioxygenase domain 0.039

rpa3724 periplasmic binding protein 0.039

rpa4019 putative branched-chain amino acid ABC transporter system substrate-binding

protein 0.038

rpa3669 putative urea short-chain amide or branched-chain amino acid uptake ABC transporter periplasmic solute-binding protein precursor 0.035

rpa4772 ornithine carbamoyltransferase 0.035

rpa1651^b possible leucine/isoleucine/valine-binding protein precursor 0.035

rpa0027 2-dehydro-3-deoxyphosphoheptonate aldolase 0.035

rpa1741^b possible branched-chain amino acid transport system substrate-binding protein 0.034

rpa2763 putative O-acetylhomoserine sulfhydrylase 0.024

rpa2193 putative ABC transporter, perplasmic binding protein, branched chain amino

acids 0.024

rpa3719 putative high-affinity branched-chain amino acid transport system ATP-binding

protein 0.023

rpa2491 N-acetylglutamate semialdehyde dehydrogenase 0.023

rpa2724 glycine hydroxymethyltransferase 0.023

rpa4813^b possible branched chain amino acid periplasmic binding protein of ABC

transporter 0.021

rpa4209 glutamine synthetase II 0.021

rpa0235 3-isopropylmalate dehydratase small subunit 0.019

rpa4179^b conserved unknown protein 0.019

rpa1283^b homoserine/homoserine lactone/threonine efflux protein 0.018 rpa4034 ABC transporter, periplasmic branched chain amino acid binding protein 0.018

rpa3060 leucine aminopeptidase 0.017

rpa4773 putative acetylornithine aminotransferase 0.016

rpa0240 3-isopropylmalate dehydratase 0.016

rpa3429^b serine acetyltransferase 0.014

rpa1415 possible branched-chain amino acid transport system substrate-binding protein 0.014

rpa2166^b conserved hypothetical protein 0.013

rpa4041 putative branched-chain amino acid ABC transport system ATP-binding protein 0.012

rpa1984 2-dehydro-3-deoxyphosphoheptonate aldolase 0.012

rpa0985^b putative branched-chain amino acid transport system substrate- binding protein 0.012 rpa1655^b possible urea/short-chain binding protein of ABC transporter 0.012

rpa2967 glutamine synthetase I 0.011

Translation, Ribosomal Structure and Biogenesis

rpa0159 ribosomal protein L27 0.159

rpa3225 50S ribosomal protein L17 0.149

rpa0160 possible acetyltransferases. 0.091

rpa3270 50S ribosomal protein L10 0.087

rpa0433 ribosomal protein S15 0.069

rpa3227 30S ribosomal protein S11 0.065

rpa3228 30S ribosomal protein S13 0.050

rpa0039 50S ribosomal protein L35 0.046

rpa1589^b 30S ribosomal protein S4 0.042

rpa3252 elongation factor Tu 0.040

rpa0051^b putative sigma-54 modulation protein 0.038

rpa0040 translation initiation factor IF-3 0.035

rpa0038^b ribosomal protein L20 0.033

rpa0493 50S ribosomal protein L28 0.032

rpa3269 50S ribosomal protein L7/L12 0.028

rpa0526^b 50S ribosomal protein L32 0.027

rpa0918 possible 50S ribosomal protein L31 0.027

rpa4328 elongation factor G, EF-G 0.026

rpa0867 Endoribonuclease L-PSP 0.024

rpa4176 ribosomal protein S21 0.024

rpa3255^b 30S ribosomal protein S12 0.024

rpa4197 50S ribosomal protein L36 0.022

rpa3129 50S ribosomal protein L33 0.018

rpa0158 putative ribosomal protein L21 0.018

rpa2768 ribosomal protein S9 0.018

rpa2922 30S ribosomal protein S2 0.016

rpa4356 putative 50S ribosomal protein L25 0.015

rpa3111 Glu-tRNA(Gln) amidotransferase subunit C 0.015

rpa4836 30S ribosomal protein S20 0.015

rpa2651^b Regulator of chromosome condensation, RCC1:Endoribonuclease L-PSP 0.015

rpa3137 Endoribonuclease L-PSP 0.013

rpa0621 putative N-formylmethionylaminoacyl-tRNA deformylase 0.013

rpa3233 ribosomal protein S5 0.013

rpa3272^b 50S ribosomal protein L1 0.013

rpa0241 50s ribosomal protein L19 0.013

rpa2777 methionyl-tRNA synthetase 0.011

Posttranslational Modification, Protein Turnover, Chaperones

rpa0889^b small heat shock protein 0.280

rpa0453 possible NifU-like domain (residues 119-187) 0.110

rpa2895 possible small heat shock protein 0.104

rpa2959 ATP-dependent protease Lon 0.101

rpa1929 htrA-like serine protease 0.098

rpa0787 putative heat shock protein (htpX) 0.096

rpa0333 heat shock protein DnaK (70) 0.091

rpa0054^b putative small heat shock protein 0.077

rpa1126 metalloprotease (cell division protein) FtsH 0.076 rpa2960 ATP-dependent Clp protease ATP binding subunit ClpX 0.074

rpa3812^b putative holocytochrome c synthase 0.046

rpa2165^b chaperonin GroES2, cpn10 0.044

rpa3491 putative protease subunit hflK 0.044

rpa2443 probable antioxidant protein 0.040

rpa4579 possible serine protease, htrA-like 0.040

rpa3147 endopeptidase Clp: ATP-binding chain A 0.033

rpa4268 peroxiredoxin-like protein 0.032

rpa0019 cytochrome-c oxidase fixN chain, heme and copper binding subunit 0.030 rpa0966 putative membrane-bound hydrogenase component hupE 0.029 rpa0017 cytochrome oxidase subunit, small membrane protein 0.026

rpa4487 DSBA oxidoreductase:Tat pathway signal 0.024

rpa1140 chaperonin GroEL1, cpn60 0.023

rpa0331 possible heat shock protein (HSP-70 COFACTOR), grpE 0.022

rpa1606 conserved unknown protein 0.022

rpa0373 thioredoxin 0.020

rpa3159 probable glutathione S-transferase 0.019

rpa2461^b Protein of unknown function UPF0075 0.018

rpa1320^b conserved hypothetical protein 0.018

rpa4069 DUF25 0.018

rpa1576 putative glutathione S-transferase 0.017

rpa3799 DUF182 0.017

rpa3490 putative hflC protein 0.016

rpa4194 osmotically inducible protein OsmC 0.015

rpa0073 thioredoxin 0.015

rpa0452 Glycoprotease (M22) metalloprotease 0.014

rpa0598 putative glutaredoxin 0.014

rpa3488 probable serine protease 0.014

rpa2720 possible glutathione-S-transferase 0.014

rpa2164^b chaperonin GroEL2, cpn60 0.013

rpa2442 putative outer membrane protein 0.012

rpa4075 thioredoxin reductase 0.012

rpa1022^b possible outer membrane protein 0.012

rpa3627^b putative glutathione peroxidase 0.012

rpa3937 putative transcriptional regulator 0.012

photosynthesis

rpa1491^b light harvesting protein B-800-850, beta chain E (antenna pigment protein, beta

chain E) (LH II-E beta) 0.556

rpa1492^b light harvesting protein B-800-850, alpha chain E (antenna pigment protein,

alpha chain E) (LH II-E alpha) 0.414

rpa4292^b light harvesting protein B-800-850, alpha chain B (antenna pigment protein,

alpha chain B) (LH II-B alpha) 0.384

rpa4291^b light harvesting protein B-800-850, beta chain B (antenna pigment protein, beta

chain B) 0.346

rpa3013^b light harvesting protein B-800-850, beta chain D (antenna pigment protein, beta

chain D) (LH II-D beta) 0.189

rpa2654^b light harvesting protein B-800-850, beta chain A (antenna pigment protein, beta

chain A) (LH II-A beta) 0.119

rpa1526^b light-harvesting complex 1 alpha chain 0.104

rpa2653^b light harvesting protein B-800-850, alpha chain A (antenna pigment protein,

alpha chain A) (LH II-A alpha) 0.092

rpa1525^b light-harvesting complex 1 beta chain 0.069

rpa3012 light harvesting protein B-800-850, alpha chain D (antenna pigment protein,

alpha chain D) (LH II-D alpha) 0.041

rpa1549 possible photosynthetic complex assembly protein 0.036 rpa1522^c bacteriochlorophyllide reductase subunit BchX 0.031

rpa1528^b photosynthetic reaction center M protein 0.027

rpa1521 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase 0.021 rpa0260 possible photosynthesis gene regulator, AppA/PpaA family 0.020

rpa1527 photosynthetic reaction center L subunit 0.018

The high expressed genes in the starved cells of R. palustris in the dark

Acc. No. definition signal %^a

Inorganic Ion Transport and Metabolism

rpa2307 possible tonB-dependent receptor precursor 0.009 rpa1259 putative cation-transporting P-type ATPase 0.009 rpa3004 potassium-transporting ATPase, A chain, KdpA 0.009 rpa3736 putative phosphate transport system substrate-binding protein 0.010

rpa1693^b superoxide dismutase 0.010

rpa2339 possible iron response transcription regulator 0.011 rpa2281 putative low-affinity phosphate transport protein 0.011

rpa4186 Integral membrane protein TerC family 0.011

rpa0099 putative oligopeptide ABC transporter (ATP-binding protein) 0.011 rpa0502 probable HlyC/CorC family of transporters with 2 CBS domains 0.011 rpa0695 PhnG protein, phosphonate metabolism, function unknown 0.011 rpa1000 Nitrogenase-associated protein:Arsenate reductase and related 0.013

rpa2195 possible exopolyphosphatase 0.013

rpa2272 conserved unknown protein 0.013

rpa3600^b bacterioferritin 0.013

rpa2043^b putative ABC transporter, periplasmic substrate-binding protein 0.013

rpa4501 phnA-like protein 0.014

rpa4635 ferrous iron transport protein B 0.014

rpa2610 aliphatic sulfonate transport ATP-binding protein, Subunit of ABC

transporter 0.015

rpa2041 ABC-transport protein, ATP-binding protein 0.015 rpa4732 possible Cation transport regulator protein 0.015

rpa4457 putative sulfide dehydrogenase 0.016

rpa2793 pH adaptation potassium efflux system phaF 0.016 rpa0691 phosphonate ABC transporter, ATP-binding component,PhnK protein 0.017

rpa2120 putative hemin binding protein 0.017

rpa2353 putative nitrogenase NifH subunit 0.018

rpa0517 putative transcriptional regulator (Fur family) 0.019

rpa1496 possible monooxygenase 0.020

rpa4636 FeoA family 0.020

rpa0688 ATP-binding component, PhnN protein, possible kinase 0.023 rpa0724 putative high-affinity nickel-transport protein 0.027 rpa1773 putative DMT superfamily multidrug-efflux transporter 0.030 rpa3002^b potassium-transporting atpase c chain, KdpC 0.062

photosynthesis

rpa1491^b light harvesting protein B-800-850, beta chain E (antenna pigment protein,

beta chain E) (LH II-E beta) 0.034

rpa1526^b light-harvesting complex 1 alpha chain 0.024

rpa3367 possible activator of photopigment and puc expression, appA-like 0.022 rpa4292^b light harvesting protein B-800-850, alpha chain B (antenna pigment protein,

alpha chain B) (LH II-B alpha) 0.020

rpa1492^b light harvesting protein B-800-850, alpha chain E (antenna pigment protein,

alpha chain E) (LH II-E alpha) 0.020

rpa2654^b light harvesting protein B-800-850, beta chain A (antenna pigment protein,

beta chain A) (LH II-A beta) 0.020

rpa4291^b light harvesting protein B-800-850, beta chain B (antenna pigment protein,

beta chain B) 0.018

rpa1528^b photosynthetic reaction center M protein 0.016

rpa3013^b light harvesting protein B-80-850, beta chain D (antenna pigment protein, beta

chain D) (LH II-D beta) 0.014

rpa1525^b light-harvesting complex 1 beta chain 0.014

rpa0522 possible activator of photopigment and puc expression 0.012 rpa2653^b light harvesting protein B-800-850, alpha chain A (antenna pigment

protein,alpha chain A) (LH II-A alpha) 0.010

Signal % means the ratio of signal intensity of each genes to total signal intensity.

The high expression genes from the top of high expression gene in the light and dark were 638 and 646, respectively, that covering 90% of total signal. Light conditions, signal intensity of genes was over 0.011%; dark conditions, signal intensity of genes was over 0.009%. The all high expression genes were Table SIII-4.

Highly gene expression were observed both in the starved cells of R. palustors CGA009 in the light or dark.

Gene was categorized to Inorganic Ion Transport and Metabolism. Other genes related

to photosynthesis were categorized to “not in COG”.

Table III-6. The high expressed genes that cording redox enzyme in starved R.

palustris CGA009 cells in the light and dark.

High expressed gene^a

definition Acc. No. Light Dark

Dehydrogenases (electron donating enzymes) Carbon monoxide dehydrogenase

(cosSML) rpa4666-4668 rpa4666, 4667

Carbon monoxide dehydrogenase rpa3802, 3803 rpa3802, 3803

Succinate dehydrogenase rpa0216 - 0219 rpa0219

Formate dehydrogenase fdsG, fdsB,

fdsA, fdsC, fdsD rpa0732-0736

Ethanol dehydrogenase (quino

hemeprotien) rpa3188

Hydrogenase (hup/hyp genes) rpa0959-0978 rpa0966 rpa0960, 0967 NADH dehydrogenase

(nuoN-nuoA) rpa2937-2952

NADH dehydrogenase

(nuoN-nuoA) rpa4252-4264 rpa4253

Thiosulfate oxidase rpa2937-2952 Oxidases (electron accepting enzymes)

Cytochrome bd ubiquinol-oxidase rpa1318, rpa1319 Cytochrome cbb3 oxidase (high

affinity oxygen) rpa0014-0019 rpa0016, 0017, 0018, 0019 Cytochrome aa3 oxidase

(coxABCEFG) rpa0831-0837 rpa0834

Quinol oxidase (qxtAB) rpa4793, rpa4794

Nitric oxide reductase (nor genes) rpa1453-1458 rpa1453, 1455 Nitrous oxide reductase noz genes rpa2060-2066

Nitrite reductase (nirK) rpa3306

Nitrite reductase (nirK) rpa4145 rpa4145

Carbon dioxide fixation

Type I RubisCo (cbbS) rpa1559, 1560 rpa1559 Type II RubisCo (cbbM) rpa4641

Nitrogen fixation and acquistion

Vanadium nitrogenase rpa1370-1380

Iron nitrogenase rpa1435-1439 rpa1437

Molybdenum nitrogenase rpa4602-4633 rpa4620

• Glutamine synthetase. glnA4 rpa0984

• Glutamine synthetase glnA rpa2967 rpa2967

• Glutamine synthetase glnAII rpa4209 rpa4209

• Glutamine synthetase gln AIII rpa1401

High expressed genes were identified as described in Table 3.

Selected genes were referenced from R. palustris CGA009 genome information (25).

DISCUSSION

Although some metabolic characteristics and energy states of the growth-arrested cells compared to that of the growing cells have been reported (8, 13, 36), it is not clear yet that the effect of energy level on the metabolism in the starved cells because the control of energy level in bacterial cells is difficult. In the present study, I performed metabolome analysis for the starved cells of purple photosynthetic bacteria that can synthesize ATP by light. I found that the metabolic profile of the starved R. palustris CGA009 cells in the light were clearly different from the starved cells incubated in the dark at time after 5 days of carbon-starvation. This is the first observation; metabolically dynamic change occurred by supplying different levels of energy under starvation conditions in bacteria.

In this study, the starved cells were subjected to the anaerobic conditions in

which little external electron accepters and donors were present, although the starved

cells in the light could synthesize ATP by photophosphorylation. The starved cells in

the dark may have obtained considerably a little energy as indicated by the low ATP

levels (Fig. I-2, Table III-2). Energy level is generally expressed by adenylate energy

charge ([(ATP) + 1/2 (ADP)]/[(ATP) + (ADP) + (AMP)]) and the energy charge affects

the balance of anabolic and catabolic reactions. High energy level promotes anabolism

rather than catabolism (2). Chapman et al. had reported about the energy charge values

of the growing cells and the growth-arrested cells in some bacteria (8). Generally, the

energy charge in the growing cells were 0.8-0.9. On the other hand, under the

growth-arrested conditions, such as nutrients limitation, energy charge decreased to a low level due to the limitation of energy source. When energy charge becomes less than 0.5, the viability of cells began to decrease. In the present study, using purple photosynthetic bacteria that can synthesize ATP by photophosphorylation, energy charge of the starved cells in the light was 0.89 and that in the dark was 0.66 at the 5th day from the beginning of the starvation. The metabolome analysis showed that in the starved cells incubated in the light the amounts of various amino acids were relatively high and the amounts of metabolites relating to the glycolytic pathway and the TCA cycle were low (Fig. III-1). These results may suggest that in the illuminated starved cells, amino acid metabolism is active to support protein biosynthesis in the present of sufficient amount of ATP. High expression of many genes related to protein turnover and high NAD

⁺

/NADH ratio (Fig. III-2b) also support this idea. It is noteworthy that it was reported that cells in the stationary phase accumulated various amino acids and protein degradation was important to survive (20, 35). Thus, a part of amino acids accumulation in the light may be due to protein degradation. While in the dark, high levels of metabolites related to the central carbon metabolism were observed. It can be suggested that macromolecule biosynthesis is repressed in the starved cells having low level of energy.

It was known that one of the responses to growth arrest was the change of inner

membrane composition to reduce membrane fluidity (31). In the present study, the ratio

of unsaturated fatty acids decreased during 5 days of the starvation both in the light and

dark. These changes may be one of the starvation responses in R. palustris CGA009. In

the illuminated starved cells, photophosphorylation may have supported fatty acid turnover and then the change of fatty acid composition became remarkable compared to that in the dark. The result suggested that starvation response in the light and dark was different depending on the energy states of the starved cells. It was known that unsaturated fatty acids of cellular membrane are converted into cyclopropyl derivatives in a growth arrested phase (30). The major component may also be a cyclopropyl fatty acid in this study but it was not identified (Fig. III-3). In future, detailed analyses of fatty acids composition including cyclopropyl fatty acids in the starved cells may contribute to understand the relationship between lipid composition of the starved cells and energy states in bacteria.

Since metabolic status in the starved cells became markedly changed by

illumination, it was expected that transcriptional status was also changed by

illumination. The starved cells both in the light and dark contained considerable amount

of mRNA. Since the half-life of bacterial mRNA is typically only ~5 min (1, 9), it was

suggested that active gene expression still occurred in the starved cells even when

energy supply was very low in the dark. Global gene expressions of the starved cells in

the light and dark were investigated and highly expressed genes were significantly

different by illumination (Fig. III-4). Many of the genes related to protein turnover were

highly expressed in the light, while mRNAs for membrane translocators of inorganic

ions were noticeable in the dark. These different patterns were probably reflected the

starvation strategy corresponding to energy status of the starved cells. In the dark, the

starved cells seemed to require translocators to maintain intracellular environments such

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 79-102)