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
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
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
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
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
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
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
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
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
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
CHAPTER I
Light-dependent survivability in purple photosynthetic bacteria under
carbon starvation conditions
5
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
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
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
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
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
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
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
(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
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
(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
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
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
(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
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
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
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
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
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
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
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
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
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
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
CHAPTER II
Species-dependent survivability in purple photosynthetic bacteria under
carbon starvation conditions
29
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
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
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
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
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
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
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
(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
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
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
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
(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"""
%$survival"
60min"
80"
0.8""
R.#palustris#
60"
0.6""
R.#sphaeroides#
40"
0.4""
R.#gela3nosus#
20"
0.2""
0"
0.0""
10min"
120min"
Fig. II-3. Effect of the osmotic pressure and heat stress on viability of starved cells.
CFUs were determined for starved cells after exposure to 0.7 M sucrose for 10, 30, 60
min (a) and 2.0 M sucrose for 10, 120 min (b), and 2.5 M NaCl for 10, 30, 60 min (c) or
10, 120 min (d) or 50°C for 10, 120 min (e) in the dark. The results are expressed as
percent of the CFUs determined after incubation for each time without exposure to the
stresses.
41
(c)$ "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
%$survival"
100"
R.#palustris"
80"
R.#sphaeroides"
60"
R.#gelatinosus"
40"
20"
0"
10min"
120min"
Fig. II-3. Continued.
42
(a)$
" 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
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
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
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
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
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
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De Philippis, R., A. Ena, M. Guastini, C. Sili, and M. Vincenzini. 1992. Factors
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Feng, Y., X. Lin, Y. Yu, and J. Zhu. 2011. Elevated ground-level O3 changes the
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Hanada, S., A. Hiraishi, K. Shimada, and K. Matsuura. 1995. Chloroflexus
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Harada, N., S. Otsuka, M. Nishiyama, and S. Matsumoto. 2003. Characteristics of
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Haruta, S. 2013. Rediscovery of the microbial world in microbial ecology.
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10. Hisada,T., K. Okamura and A. Hiraishi. 2007. Isolation and characterization of
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11. Iacobellis, N.S., and J.E. Devay. 1986. Long-term storage of plant-pathogenic
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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
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15. Imhoff, J.F. 2005. Genus incertae sedis XV, Rubrivivax Willems, Gillis, and De
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Rhodopseudomonas palustris SP5212. World J. Microb. Biot. 21:765-769.
19. Okamura, K., K. Takata and A. Hiraishi. 2009. Intrageneric relationships of
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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
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21. Oda, Y., W. Wanders, L.A. Huisman, W.G. Meijer, J.C. Gottschal and L.J. Forney.
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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.
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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
CHAPTER III
Effect of light on metabolomic and transcriptomic profile of starved cells in
a purple photosynthetic bacterium Rhodopseudomonas palustris
53
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
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
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
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
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
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
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
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 MgCl2], 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 Na2EDTA;
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
61
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.
(iv) Analysis of microarray data.
The expression value of each gene was determined by calculating the average
of signal values of three probes. 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. Those genes were categorized to Clusters of Orthologous Groups (COG)
classifications using CyanoBase
(http://genome.microbedb.jp/cyanobase/Rhodopseudomonas).
62
RESULTS
Effect of light on metabolic profile of starved cells.
Cellular metabolites examined in this study were 108 kinds of molecules
expected to be important and abundant in general cells. Carbon starved cells of
anoxygenic heterotrophic photosynthetic bacteria, R. palustris CGA009, were used for
the metabolite study. Un-illuminated starved cells were able to maintain colony-forming
viability for 5 days of starvation, and then colony-forming viability was gradually
decreased (Fig. I-2a). Whereas illuminated cells were survived more than 30 days with
little loss of viability (Fig. I-2a). I examined metabolites in 5 day starved cells in both
the light and dark where viabilities of both cells were maintained.
78 and 80 metabolites in the light and dark, respectively, were identified and
quantified by CE-MS analysis (Table III-2, Table S III-2). Total amounts of metabolites
obtained in this study were slightly different between the cells in the light and dark; that
were 7086 and 10479 pmol OD660
-1
mL-1, respectively (Table III-1). Significant
difference in the amounts of metabolites between the cells in the light and dark were
observed in almost all target metabolic pathways in this study including the glycolytic
pathway, the TCA cycle, the pentose-phosphate cycle, the amino acids metabolism and
the nucleic acids metabolism. Metabolic profiles of marked changes (over 2-fold) are
shown in Table III-2. In metabolites accumulated more than 2 times in the light
compared to the dark, the high amount of ATP was detected in the light as expected.
Other abundant metabolites in the light were 12 species of amino acids (Gln, Pro, Arg,
63
Asn, Thr, Leu, Val, Ile, Phe, Tyr, Trp, Ser). On the other hand, more than 2-times
accumulated metabolites in the dark were prominent in the metabolites related to the
glycolytic pathway, the pentose-phosphate cycle and the TCA cycle. Fumarate was
accumulated 3.5-fold in the starved cells in the dark and small amount of fumarate was
also observed in the supernatant of the culture in the dark but not in the light (Table
SIII-3). Fig. III-1 summarizes the quantitative ratio of metabolites on a map of central
metabolism and amino acids; it is noteworthy that orange-marked metabolites which
were observed more than 2 times in the light were mostly amino acids, blue-marked
metabolites which were more abundant in the dark were mostly those in the sugar
metabolism and TCA cycle. Exceptional amino acids were glutamic acid that has a key
role of amino acids biosynthesis and lysine that has the extra amino residue. These
amino acids were more accumulated in the dark. Especially lysine was markedly
accumulated in the dark; 287 and 2087 pmol OD660-1 mL-1, in the light and dark,
respectively. In addition, marked accumulation of intermediates of nucleotide
metabolism was observed; UTP (L/D was 52) was markedly accumulated in the light
and IMP was detected only in the dark.
64
Table III-1. Concentration of total metabolites in R. palustris CGA009 under carbon
starvation conditions in the light and the dark.
a
Incubation conditions
Total metabolites (pmol OD660-1mL-1)b
Darka
7086
Lighta
10479
Metabolites were extracted from starved cells maintained in the light or dark
conditions for 5days.
b
Total metabolites are the sum of all the metabolites detected by CE-MS.
65
Table III-2. The concentration of metabolites of starved R. palustris CGA009 cells in
the light and dark.
Light/Dark ratio >2.0
Concentration of metabolite
(pmol OD660-1 mL-1)
a
Metabolite
Dark
Light
cAMP
N.D.
0.5
CoA_divalent
N.D.
5.5
Malonyl CoA_divalent
N.D.
1.9
S-Adenosylmethionine
N.D.
31
Homoserine
1.4
75
UTP
72
1,399
Glutathione (GSSG)_divalent
3.7
59
Acetyl CoA_divalent
11
180
dTTP
2.8
42
Putrescine
105
1,210
Gln
3.0
31
Asn
2.2
22
Ile
2.5
21
dATP
2.7
21
Phe
2.3
16
cGMP
0.3
2.1
Tyr
1.6
9.4
Pro
14
78
NADP+
68
293
Trp
1.5
6.1
Citrulline
1.2
4.9
ATP
984
3,715
UDP
68
244
dTDP
1.7
5.5
Leu
6.5
19
Glyceraldehyde 3-phosphate
1.9
4.8
Ser
14
34
Arg
94
219
Val
8.4
19
Thr
39
80
66
Light / Dark
1<
1<
1<
1<
52.0
19.0
16.0
16.0
15.0
12.0
10.0
10.0
8.3
7.8
7.1
6.5
6.0
5.5
4.3
4.1
4.0
3.8
3.6
3.2
2.9
2.5
2.5
2.3
2.3
2.1
Metabolitea
Choline
Glucose 6-phosphate
Adenosine
2-Oxoisovaleric acid
Sedoheptulose 7-phosphate
Fumaric acid
Glycerol 3-phosphate
Glu
Ribose 5-phosphate
Ornithine
3-Phosphoglyceric acid
CMP
Malic acid
Fructose 1,6-diphosphate
Uridine
2-Phosphoglyceric acid
CDP
Lys
Ribulose 5-phosphate
Phosphoenolpyruvic acid
GDP
GMP
Guanine
Cytosine
Glyoxylic acid
Erythrose 4-phosphate
IMP
β-Ala
Cytidine
Guanosine
a
Light/Dark ratio <0.5
Concentration of metabolite
(pmol OD660-1 mL-1)
Dark
Light
40
20
54
25
10
3.8
1.6
0.5
46
13
13
3.7
18
5.0
298
78
3.8
0.9
16
3.8
76
18
113
24
61
13
15
2.9
26
4.7
17
3.0
108
17
2,087
287
22
2.7
55
6.3
190
20
70
6.6
46
3.1
8.2
0.3
3.4
N.D.
4.4
N.D.
28
N.D.
5.7
N.D.
27
N.D.
48
N.D.
Light / Dark
0.49
0.46
0.38
0.31
0.28
0.28
0.28
0.26
0.24
0.24
0.23
0.21
0.21
0.20
0.18
0.18
0.16
0.14
0.12
0.11
0.10
0.09
0.07
0.04
<1
<1
<1
<1
<1
<1
Metabolites were extracted from starved cells of R. palustris CGA009 maintained in
the light or dark conditions for 5 days and analyzed by CE-MS.
N.D.; not detected.
Table III-2. Continued.
67
Fig. III-1. Effect of light on the central metabolism and the amino acids metabolism in
the starved R. palustris CGA009 cells. The data of each metabolite level from Table
III-2 and Table SIII-2 are inserted to this figure. Orange-marked metabolites were
observed more than 2 times in the light; blue-marked metabolites were observed more
than 2 times in the dark; black-marked metabolites were observed similar concentration
both in the light and dark; gray-marked metabolites were not detected both in the light
and dark.
68
Redox status of starved cells in the light and dark.
The nicotinamide adenine dinucleotide is an essential cofactor that is required
for various biosynthesis, energy metabolism and redox balancing. To understand the
effect of light on redox status in the starved cells, I measured the amount of both
oxidized and reduced form of nicotinamide adenine dinucleotide in the growing cells
and the starved cells in the light and dark. Fig. III-2a shows the total amount of
nicotinamide adenine dinucleotide in the growing cells and the starved cells of R.
palustris CGA009. Total pool size of nicotinamide adenine dinucleotide in growing
cells was bigger than that in the starved cell (more than 2-fold) and the starved cells in
the dark showed lowest level of total pool of nicotinamide adenine dinucleotide in the
three preparations of cells. Next, I calculated NAD+/NADH ratios in the growing cells
and the starved cells in the light and dark (Fig. III-2b). The starved cells in the dark and
growing cells showed similar NAD+/NADH ratios; 13.4 and 17.4 in the starved cells in
the dark and the growing cells, respectively. It was suggested that the starved cells of R.
palustris CGA009 in the dark probably maintained the redox states until 5 days
starvation, although the total amount of NAD decreased to a low level. The starved cells
in the light had more than 2.3-fold higher molecular NAD+/NADH ratio compared to
both the starved cells in the dark and the growing cells. This oxidative state of starved
cells in the light might be due to biosynthetical usage of the reduced nicotinamide
adenine dinucleotide.
69
(a)
Total nicotinamide adenine
dinucleotide (nM/OD660)
900
800
700
600
500
400
300
200
100
0
Light 5 day
Dark 5 day
Exponential
growing
50
(b)
NAD+/NADH ratio
40
30
20
10
0
Light 5 day
Dark 5 day
Exponential
growing
Fig. III-2. Effect of growth phase and light on the amount of nicotinamide adenine
dinucleotides of the R. palustris CGA009 cells. NAD+ and NADH were extracted form
the starved cells and the growing cells and total concentration of nicotinamide adenine
dinucleotides (a) and NAD+/NADH ratio (b) were calculated. The starved cells were
incubated in the light and dark for 5 days starvation. The growing cells were grown to
log-phase in culture containing 0.5% sodium succinate. Data are presented as the means
of three independent cultures, and error bars represent standard deviations.
70
Change in the composition of membrane fatty aids in starved cells in the light and dark.
In some bacteria, the composition of membrane fatty acids changes to less fluid
states responding to the nutrient starvation or growth arrest (30, 31). The membrane
fatty acid composition of the starved cells of R. palustris CGA009 was obtained under
the light and dark conditions. In a previous report, fatty acids of hexadecanoic (palmitic)
acid (C16:0), hexadecenoic (palmitoleic) acid (C16:1), octadecanoic (stearic) acid
(C18:0) and octadecenoic (oleic or vaccenic) acids (C18:1) were reported in R. palustris
and the presence of C14:0 and C14:1 were suggested (21). In the present study, four
peaks were identified as C18:1, C18:0, C16:1 and C16:0 (Table III-3). Retention time of
16.13 min may be due to a cyclic fatty acid such as a cyclopropyl fatty acid but it was
not identified in this study (Fig. III-3). To compare the differences in membrane fatty
acid composition of the starved cells of R. palustris CGA009, the total saturated fatty
acids and the total unsaturated fatty acids were estimated. The ratio of the unsaturated
fatty acids was used as an indirect indicator of the membrane fluidity. It has been
previously reported that membranes with high ratio of unsaturated fatty acids show a
high fluidity (7). The ratio of unsaturated fatty acids of the cells at the time of beginning
of starvation was 83.5%. After 5 days of starvation, the ratio of the unsaturated fatty
acids in the starved cells was decreased to 73.5% and 81.7% in the light and dark,
respectively (Table III-3). The decrease of the proportion of the unsaturated fatty acids
was mainly caused by the decrease of unsaturated fatty acids in the dark (saturated fatty
acids were increased from 0.9 µg mL-1 to 0.97 µg mL-1 and unsaturated fatty acids were
decreased from 4.52 µg mL-1 to 4.31 µg mL-1), while change in both the proportion of
71
unsaturated fatty acid and saturated fatty acid contributed the decrease of the ratio of
unsaturated fatty acids in the light (saturated fatty acids from 0.9 µg mL-1 were
increased to 1.35 µg mL-1 and unsaturated fatty acids were decreased from 4.52 µg mL-1
to 3.73 µg mL-1). These results may suggest that membrane fluidity of the starved R.
palustris CGA009 cells was decreased by incubation under starvation conditions and
light enhanced the decrease of membrane fluidity.
72
(a)
Abundance
C18:1
C16:0C16:1 C18:0
Time (min)
(b)
Abundance
C18:1
C16:0 C16:1 C18:0
Time (min)
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.
73
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)a
Fatty acids
Beginning
of starvationb
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 %c
83.5
a
81.7
73.5
Metabolites were extracted from starved culture maintained in the light or dark
conditions for 5days.
b
“Beginning of starvation” was defined as the time when the growth completely
stopped.
c
Unsaturation degree % means the ratio of sum of C16:1 and C18:1 to total fatty acids.
74
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
OD660-1. 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 OD660-1, 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.
75
Table III-4. RNA levels of starved cells and growing cells in R. palustris CGA009.
Concentration of RNA (µg OD660-1 mL-1)a
a
Conditions
Total RNAa
rRNAb
mRNAb
tRNAb
Dark 5day
1.09
0.80
0.26
0.02
Light 5day
Exponential
growth phase
1.95
1.03
0.78
0.14
4.96
-
-
-
Total RNA were extracted from the starved cells and the growing cells, as described at
Materials and Methods.
b
Concentrations of rRNA, mRNA, tRNA are calculated using results of microarray
analysis. rRNA; rpa_RNA50-52, 57, 58. tRNA; rpa_RNA1-43, 46-49.
76
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
77
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
78
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.
79
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
80
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
81
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.
82
Table III-5. The high expressed genes categorized selected COGs in starved R.
palustris CGA009 cells in the light and dark.
Acc. No.
rpa3033
rpa0870
rpa4020b
rpa3810
rpa1789
rpa2966
rpa3093
rpa3725b
rpa2046
rpa2446
rpa1798b
rpa0230
rpa0668
rpa2503
rpa4807
rpa3297b
rpa4331
rpa0557
rpa1664
rpa3724
rpa4019
rpa3669
rpa4772
rpa1651b
rpa0027
rpa1741b
rpa2763
rpa2193
rpa3719
rpa2491
rpa2724
rpa4813b
rpa4209
rpa0235
rpa4179b
rpa1283b
rpa4034
rpa3060
rpa4773
rpa0240
rpa3429b
rpa1415
rpa2166b
rpa4041
rpa1984
rpa0985b
rpa1655b
rpa2967
rpa0159
The high expressed genes in the starved cells of R. palustris in the light
definition
Amino Acid Transport and Metabolism
possible acetylornitine deacetylase
putative ornithine decarboxylase
possible branched-chain amino acid transport system permease protein
putative periplasmic binding protein of ABC transporter
putative branched-chain amino acid transport system substrate-binding protein
nitrogen regulatory protein P-II
possible urea/short-chain binding protein of ABC transporter
possible leucine/isoleucine/valine-binding protein precursor
2-isopropylmalate synthase
putative aminotransferase
putative periplasmic binding protein for ABC transporter for branched chain
amino acids
aspartate-semialdehyde dehydrogenase
putative ABC transporter subunit, substrate-binding component
possible aminotransferase
possible branched-chain amino acid transport system substrate-binding protein
possible branched-chain amino acid transport system substrate-binding protein
aspartate aminotransferase A
cysteine synthase, cytosolic O-acetylserine(thiol)lyase
Glyoxalase/Bleomycin resistance protein/dioxygenase domain
periplasmic binding protein
putative branched-chain amino acid ABC transporter system substrate-binding
protein
putative urea short-chain amide or branched-chain amino acid uptake ABC
transporter periplasmic solute-binding protein precursor
ornithine carbamoyltransferase
possible leucine/isoleucine/valine-binding protein precursor
2-dehydro-3-deoxyphosphoheptonate aldolase
possible branched-chain amino acid transport system substrate-binding protein
putative O-acetylhomoserine sulfhydrylase
putative ABC transporter, perplasmic binding protein, branched chain amino
acids
putative high-affinity branched-chain amino acid transport system ATP-binding
protein
N-acetylglutamate semialdehyde dehydrogenase
glycine hydroxymethyltransferase
possible branched chain amino acid periplasmic binding protein of ABC
transporter
glutamine synthetase II
3-isopropylmalate dehydratase small subunit
conserved unknown protein
homoserine/homoserine lactone/threonine efflux protein
ABC transporter, periplasmic branched chain amino acid binding protein
leucine aminopeptidase
putative acetylornithine aminotransferase
3-isopropylmalate dehydratase
serine acetyltransferase
possible branched-chain amino acid transport system substrate-binding protein
conserved hypothetical protein
putative branched-chain amino acid ABC transport system ATP-binding protein
2-dehydro-3-deoxyphosphoheptonate aldolase
putative branched-chain amino acid transport system substrate- binding protein
possible urea/short-chain binding protein of ABC transporter
glutamine synthetase I
Translation, Ribosomal Structure and Biogenesis
ribosomal protein L27
83
signal %a
1.020
0.908
0.457
0.163
0.106
0.103
0.092
0.081
0.071
0.069
0.058
0.055
0.055
0.047
0.042
0.041
0.040
0.039
0.039
0.039
0.038
0.035
0.035
0.035
0.035
0.034
0.024
0.024
0.023
0.023
0.023
0.021
0.021
0.019
0.019
0.018
0.018
0.017
0.016
0.016
0.014
0.014
0.013
0.012
0.012
0.012
0.012
0.011
0.159
rpa3225
rpa0160
rpa3270
rpa0433
rpa3227
rpa3228
rpa0039
rpa1589b
rpa3252
rpa0051b
rpa0040
rpa0038b
rpa0493
rpa3269
rpa0526b
rpa0918
rpa4328
rpa0867
rpa4176
rpa3255b
rpa4197
rpa3129
rpa0158
rpa2768
rpa2922
rpa4356
rpa3111
rpa4836
rpa2651b
rpa3137
rpa0621
rpa3233
rpa3272b
rpa0241
rpa2777
rpa0889b
rpa0453
rpa2895
rpa2959
rpa1929
rpa0787
rpa0333
rpa0054b
rpa1126
rpa2960
rpa3812b
rpa2165b
rpa3491
rpa2443
rpa4579
rpa3147
rpa4268
rpa0019
rpa0966
rpa0017
rpa4487
rpa1140
rpa0331
rpa1606
rpa0373
rpa3159
rpa2461b
rpa1320b
rpa4069
50S ribosomal protein L17
possible acetyltransferases.
50S ribosomal protein L10
ribosomal protein S15
30S ribosomal protein S11
30S ribosomal protein S13
50S ribosomal protein L35
30S ribosomal protein S4
elongation factor Tu
putative sigma-54 modulation protein
translation initiation factor IF-3
ribosomal protein L20
50S ribosomal protein L28
50S ribosomal protein L7/L12
50S ribosomal protein L32
possible 50S ribosomal protein L31
elongation factor G, EF-G
Endoribonuclease L-PSP
ribosomal protein S21
30S ribosomal protein S12
50S ribosomal protein L36
50S ribosomal protein L33
putative ribosomal protein L21
ribosomal protein S9
30S ribosomal protein S2
putative 50S ribosomal protein L25
Glu-tRNA(Gln) amidotransferase subunit C
30S ribosomal protein S20
Regulator of chromosome condensation, RCC1:Endoribonuclease L-PSP
Endoribonuclease L-PSP
putative N-formylmethionylaminoacyl-tRNA deformylase
ribosomal protein S5
50S ribosomal protein L1
50s ribosomal protein L19
methionyl-tRNA synthetase
Posttranslational Modification, Protein Turnover, Chaperones
small heat shock protein
possible NifU-like domain (residues 119-187)
possible small heat shock protein
ATP-dependent protease Lon
htrA-like serine protease
putative heat shock protein (htpX)
heat shock protein DnaK (70)
putative small heat shock protein
metalloprotease (cell division protein) FtsH
ATP-dependent Clp protease ATP binding subunit ClpX
putative holocytochrome c synthase
chaperonin GroES2, cpn10
putative protease subunit hflK
probable antioxidant protein
possible serine protease, htrA-like
endopeptidase Clp: ATP-binding chain A
peroxiredoxin-like protein
cytochrome-c oxidase fixN chain, heme and copper binding subunit
putative membrane-bound hydrogenase component hupE
cytochrome oxidase subunit, small membrane protein
DSBA oxidoreductase:Tat pathway signal
chaperonin GroEL1, cpn60
possible heat shock protein (HSP-70 COFACTOR), grpE
conserved unknown protein
thioredoxin
probable glutathione S-transferase
Protein of unknown function UPF0075
conserved hypothetical protein
DUF25
84
0.149
0.091
0.087
0.069
0.065
0.050
0.046
0.042
0.040
0.038
0.035
0.033
0.032
0.028
0.027
0.027
0.026
0.024
0.024
0.024
0.022
0.018
0.018
0.018
0.016
0.015
0.015
0.015
0.015
0.013
0.013
0.013
0.013
0.013
0.011
0.280
0.110
0.104
0.101
0.098
0.096
0.091
0.077
0.076
0.074
0.046
0.044
0.044
0.040
0.040
0.033
0.032
0.030
0.029
0.026
0.024
0.023
0.022
0.022
0.020
0.019
0.018
0.018
0.018
rpa1576
rpa3799
rpa3490
rpa4194
rpa0073
rpa0452
rpa0598
rpa3488
rpa2720
rpa2164b
rpa2442
rpa4075
rpa1022b
rpa3627b
rpa3937
rpa1491b
rpa1492
b
rpa4292
b
rpa4291
b
rpa3013
b
rpa2654
b
rpa1526b
rpa2653
b
rpa1525
b
rpa3012
rpa1549
rpa1522c
rpa1528b
rpa1521
rpa0260
rpa1527
Acc. No.
rpa2307
rpa1259
rpa3004
rpa3736
rpa1693b
rpa2339
rpa2281
rpa4186
rpa0099
rpa0502
rpa0695
rpa1000
rpa2195
rpa2272
rpa3600b
rpa2043b
putative glutathione S-transferase
DUF182
putative hflC protein
osmotically inducible protein OsmC
thioredoxin
Glycoprotease (M22) metalloprotease
putative glutaredoxin
probable serine protease
possible glutathione-S-transferase
chaperonin GroEL2, cpn60
putative outer membrane protein
thioredoxin reductase
possible outer membrane protein
putative glutathione peroxidase
putative transcriptional regulator
photosynthesis
light harvesting protein B-800-850, beta chain E (antenna pigment protein, beta
chain E) (LH II-E beta)
light harvesting protein B-800-850, alpha chain E (antenna pigment protein,
alpha chain E) (LH II-E alpha)
light harvesting protein B-800-850, alpha chain B (antenna pigment protein,
alpha chain B) (LH II-B alpha)
light harvesting protein B-800-850, beta chain B (antenna pigment protein, beta
chain B)
light harvesting protein B-800-850, beta chain D (antenna pigment protein, beta
chain D) (LH II-D beta)
light harvesting protein B-800-850, beta chain A (antenna pigment protein, beta
chain A) (LH II-A beta)
light-harvesting complex 1 alpha chain
light harvesting protein B-800-850, alpha chain A (antenna pigment protein,
alpha chain A) (LH II-A alpha)
light-harvesting complex 1 beta chain
light harvesting protein B-800-850, alpha chain D (antenna pigment protein,
alpha chain D) (LH II-D alpha)
possible photosynthetic complex assembly protein
bacteriochlorophyllide reductase subunit BchX
photosynthetic reaction center M protein
2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase
possible photosynthesis gene regulator, AppA/PpaA family
photosynthetic reaction center L subunit
The high expressed genes in the starved cells of R. palustris in the dark
definition
Inorganic Ion Transport and Metabolism
possible tonB-dependent receptor precursor
putative cation-transporting P-type ATPase
potassium-transporting ATPase, A chain, KdpA
putative phosphate transport system substrate-binding protein
superoxide dismutase
possible iron response transcription regulator
putative low-affinity phosphate transport protein
Integral membrane protein TerC family
putative oligopeptide ABC transporter (ATP-binding protein)
probable HlyC/CorC family of transporters with 2 CBS domains
PhnG protein, phosphonate metabolism, function unknown
Nitrogenase-associated protein:Arsenate reductase and related
possible exopolyphosphatase
conserved unknown protein
bacterioferritin
putative ABC transporter, periplasmic substrate-binding protein
85
0.017
0.017
0.016
0.015
0.015
0.014
0.014
0.014
0.014
0.013
0.012
0.012
0.012
0.012
0.012
0.556
0.414
0.384
0.346
0.189
0.119
0.104
0.092
0.069
0.041
0.036
0.031
0.027
0.021
0.020
0.018
signal %a
0.009
0.009
0.009
0.010
0.010
0.011
0.011
0.011
0.011
0.011
0.011
0.013
0.013
0.013
0.013
0.013
rpa4501
rpa4635
rpa2610
rpa2041
rpa4732
rpa4457
rpa2793
rpa0691
rpa2120
rpa2353
rpa0517
rpa1496
rpa4636
rpa0688
rpa0724
rpa1773
rpa3002b
rpa1491b
rpa1526
rpa3367
b
rpa4292b
rpa1492b
rpa2654b
rpa4291b
rpa1528
b
rpa3013
b
rpa1525b
rpa0522
rpa2653b
a
phnA-like protein
ferrous iron transport protein B
aliphatic sulfonate transport ATP-binding protein, Subunit of ABC
transporter
ABC-transport protein, ATP-binding protein
possible Cation transport regulator protein
putative sulfide dehydrogenase
pH adaptation potassium efflux system phaF
phosphonate ABC transporter, ATP-binding component,PhnK protein
putative hemin binding protein
putative nitrogenase NifH subunit
putative transcriptional regulator (Fur family)
possible monooxygenase
FeoA family
ATP-binding component, PhnN protein, possible kinase
putative high-affinity nickel-transport protein
putative DMT superfamily multidrug-efflux transporter
potassium-transporting atpase c chain, KdpC
photosynthesis
light harvesting protein B-800-850, beta chain E (antenna pigment protein,
beta chain E) (LH II-E beta)
light-harvesting complex 1 alpha chain
possible activator of photopigment and puc expression, appA-like
light harvesting protein B-800-850, alpha chain B (antenna pigment protein,
alpha chain B) (LH II-B alpha)
light harvesting protein B-800-850, alpha chain E (antenna pigment protein,
alpha chain E) (LH II-E alpha)
light harvesting protein B-800-850, beta chain A (antenna pigment protein,
beta chain A) (LH II-A beta)
light harvesting protein B-800-850, beta chain B (antenna pigment protein,
beta chain B)
photosynthetic reaction center M protein
light harvesting protein B-80-850, beta chain D (antenna pigment protein, beta
chain D) (LH II-D beta)
light-harvesting complex 1 beta chain
possible activator of photopigment and puc expression
light harvesting protein B-800-850, alpha chain A (antenna pigment
protein,alpha chain A) (LH II-A alpha)
0.014
0.014
0.015
0.015
0.015
0.016
0.016
0.017
0.017
0.018
0.019
0.020
0.020
0.023
0.027
0.030
0.062
0.034
0.024
0.022
0.020
0.020
0.020
0.018
0.016
0.014
0.014
0.012
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.
b
Highly gene expression were observed both in the starved cells of R. palustors
CGA009 in the light or dark.
c
Gene was categorized to Inorganic Ion Transport and Metabolism. Other genes related
to photosynthesis were categorized to “not in COG”.
86
Table III-6. The high expressed genes that cording redox enzyme in starved R.
palustris CGA009 cells in the light and dark.
definition
Acc. No.
Dehydrogenases (electron donating enzymes)
Carbon monoxide dehydrogenase
rpa4666-4668
(cosSML)
Carbon monoxide dehydrogenase
rpa3802, 3803
Succinate dehydrogenase
rpa0216 - 0219
Formate dehydrogenase fdsG, fdsB,
rpa0732-0736
fdsA, fdsC, fdsD
Ethanol dehydrogenase (quino
rpa3188
hemeprotien)
Hydrogenase (hup/hyp genes)
rpa0959-0978
NADH dehydrogenase
rpa2937-2952
(nuoN-nuoA)
NADH dehydrogenase
rpa4252-4264
(nuoN-nuoA)
Thiosulfate oxidase
rpa2937-2952
Oxidases (electron accepting enzymes)
Cytochrome bd ubiquinol-oxidase
rpa1318, rpa1319
Cytochrome cbb3 oxidase (high
rpa0014-0019
affinity oxygen)
Cytochrome aa3 oxidase
rpa0831-0837
(coxABCEFG)
Quinol oxidase (qxtAB)
rpa4793, rpa4794
Nitric oxide reductase (nor genes)
rpa1453-1458
Nitrous oxide reductase noz genes
rpa2060-2066
Nitrite reductase (nirK)
rpa3306
Nitrite reductase (nirK)
rpa4145
Carbon dioxide fixation
Type I RubisCo (cbbS)
rpa1559, 1560
Type II RubisCo (cbbM)
rpa4641
Nitrogen fixation and acquistion
Vanadium nitrogenase
rpa1370-1380
Iron nitrogenase
rpa1435-1439
Molybdenum nitrogenase
rpa4602-4633
• Glutamine synthetase. glnA4
rpa0984
• Glutamine synthetase glnA
rpa2967
• Glutamine synthetase glnAII
rpa4209
• Glutamine synthetase gln AIII
rpa1401
a
Light
High expressed genea
Dark
rpa4666, 4667
rpa3802, 3803
rpa0219
rpa0966
rpa0960, 0967
rpa4253
rpa0016, 0017,
0018, 0019
rpa0834
rpa1453, 1455
rpa4145
rpa1559
rpa1437
rpa4620
rpa2967
rpa4209
High expressed genes were identified as described in Table 3.
Selected genes were referenced from R. palustris CGA009 genome information (25).
87
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
88
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
89
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
90
as ion balance.
The regulation mechanism of many genes in the starved cells of R. palustris
CGA009 by light was unclear. In a previous study, proteomic responses to the
starvation and light have been examined in a bacteriochlorophyll-containing aerobic
bacterium, Roseobacter litoralis OCh149 (50). Their study also indicated that light
induced marked changes in protein composition in the cells under carbon-limiting
conditions, although the mechanism of the light effect was not known and how the
physiological change corresponding to energy states affected on gene regulation was
also unclear. R. palustris CGA009 has photoreceptor-encoding genes such as
bacteriophytochrome and cryptochrome-like genes. In the present study, the starved
cells were illuminated by a light above 750 nm wavelengths. In this wavelength range,
bacteriphytochrome is possible to play a part of gene regulation process. However, it
was not clear whether bacteriophytochrome, other than the energy level shown in this
study, contributes to global gene regulation including starvation survival response genes
or not. In general, genes related to metabolism are tightly regulated by various
transcriptional regulators, some of which may sense cellular redox states and energy
states (12, 40). In some species of purple photosynthetic bacteria, it was known that the
RegB/RegA two-component signaling system that work in gene regulation response to
redox changes in the photosynthetic and respiratory electron transport chains (23). In
addition, it was reported that energy states affected the gene regulation under starvation
(4, 46, 49). Because of the illumination wavelength used in this study, it is possible that
most of the starvation responding genes was probably regulated by energy or redox
91
status.
In the illuminated starved cells, many genes related to stress resistance and
some regulator genes (ecfG, CtrA and FixK) were highly expressed. In Escherichia coli,
RpoS which is a master regulator of the general stress response was extensively studied,
but RpoS orthologs are absent in the alpha subgroup of Proteobacteria (39). Recently it
was reported that a sigma factor EcfG subfamily was shown to control the general stress
response in some species of the alpha subgroup of Proteobacteria. EcfG activity is
controlled by the anti-sigma factor NepR and the anti-anti-sigma factor PhyR. In
addition it was reported that CtrA and FixK regulators related to the gene expression
under carbon starvation conditions in Caulobacter crescentus (6). It may be possible
that those regulators in alpha Proteobacteria relate to the high expression of genes in
the starved cells of R. palustris CGA009 under the light conditions.
Energy and redox status are known to directly relate to the regulation of
metabolism. In R. palustris, carbon fixation reaction and hydrogen production by
nitrogenase were important to maintain the redox homeostasis in the growing cells (28,
29). In this study, genes for a part of these enzyme complexes were shown to be
expressed under carbon starvation both in the light and dark (Table III-6). Genes for
acetate production and carbon monoxide dehydrogenase that may work as redox
enzymes were also expressed in the light. The cells in the light continuously drive the
cyclic electron transfer system that supplies electrons to quinone pool and is required to
balance the redox status. Thus it was expected that enzymes related to redox reactions
have important role to maintain cellular metabolism not only in the growing cells but
92
also in the starved cells. The presence of a small amount of fumarate in the supernatant
of un-illuminated starved cells might also indicate the necessity of redox balancing even
in the dark (Table SIII-3).
The carbon and nitrogen ratio is also known to relate to the regulation of
metabolism in bacteria (14, 38). Marked accumulation of lysine was observed in the
starved cells in the dark (Table III-2). In the starved conditions in this study, carbon
source were depleted but the nitrogen source as ammonium was rich in the culture
medium. Since lysine have two amino groups, it was possible that lysine may be
accumulated as a pool of amino group. In addition, high concentration of UTP in the
light and high concentration of IMP in the dark were observed in the starved cells
(Table III-2). UTP is an intermediate of the pyrimidine metabolism and synthesized
from UDP that is in the first step of the pyrimidine metabolism. IMP is an intermediate
of the purine metabolism and is in the first step of the purine metabolism. The high
levels of UTP and IMP may be due to the growth arrest because nucleotide biosynthesis
(replicated DNA biosynthesis) was not needed in non-growing conditions. Difference in
nucleotide species accumulated in illuminated and un-illuminated starved cells may
reflect balancing of macromolecular turnover such as sugar and nitrogen compound;
UMP that is a precursor of UTP is synthesized from aspartic acid and carbamoyl
phosphate. IMP is synthesized from 5-phosphoribosyl 1-pyrophosphate (a sugar group)
and glutamine. In the dark, IMP might be accumulated in the cells rather than UTP due
to sugar group was rich in un-illuminated cells.
93
The studies described in Chapter 1 suggested that energy production by
photosynthesis in purple bacteria promoted the survivability under starvation conditions.
In this Chapter, it was observed that illumination significantly affected the metabolic
profile and gene expression of the starved cells. Although the relationship between
metabolic phenotype and viability has not been clarified, it was suggest that the
growth-arrested purple phototrophic bacteria utilized ATP to support macromolecule
turnover and then adapted to nutrients limiting conditions properly. It is likely that
energy production is essential for growth-arrested cells for long-range survival. On the
other hand, even under the anaerobic dark conditions, some energy is provided for the
biosynthesis of maintaining proteins. Survival and death process of bacterial cells may
be really complex and understanding of contribution of energy for survival during
starvation needs further studies. The present work that showed metabolic and
transcriptional characteristics of starved cells having different energy status was a good
start for the understanding of relationship between bacterial survival and cellular energy
status.
94
SUPPLEMENTAL MATERIALS
Fig. SIII-1. RNA integrity measured by the Agilent 2100 bioanalyser.
Electropherograms (Bioanalyzer, Agilent) of total RNA of starved R. palustris CGA009
cells in the light (a) and total RNA of starved cells in R. palustris CGA009 in the dark
(b). Sharp peaks representing 16S and 23S rRNA demonstrate good quality (solid lines).
95
Table SIII-1. Quality of RNA for microarray analysis.
Category of quality check
Light 5 day
Dark 5 day
RNA concentration (ng µL-1)
87.6
97.0
rRNA Ratio (23S/16S)
1.11
1.08
3.6
3.4
0.6
0.7
166.7
205.9
Quality of RNA checked by Bioanalyzer
a
Quality of labeled cDNA checked by NanoDrop
cDNA (ng µL-1) b
-1
Cy3 (pmol µL )
Cy3 incorporation rate (pmol/Cy3 per µg cDNA)
c
a
The value was calculated using same RNA to Fig. SIII-1.
b
The recommended value of cDNA concentration is over 13ng µL-1.
c
The recommended value of Cy3 incorporation rate is over 40 pmol/Cy3 per µg cDNA.
96
Table SIII-2. A list of metabolites which concentration was similar in both
conditions or was below detection limit.
Metabolite
NAD+
Gly
UMP
Glucose 1-phosphate
His
dCTP
Citric acid
GABA
Succinic acid
2-Hydroxybutyric acid
PRPP
3-Hydroxybutyric acid
Lactic acid
Adenine
ADP
Fructose 6-phosphate
CTP
Ala
GTP
AMP
dTMP
Tyramine
Dihydroxyacetone
phosphate
Glycolic acid
Pyruvic acid
2-Oxoglutaric acid
cis-Aconitic acid
Isocitric acid
Gluconic acid
6-Phosphogluconic acid
Sarcosine
N,N-Dimethylglycine
Creatinine
Betaine
Betaine aldehyde_+H2O
Cys
Hydroxyproline
Creatine
Metabolite concentration (pmol Comparative
OD660-1 mL-1)
Analysis
Dark
Light
Light / Dark
318
610
1.92
48
87
1.83
49
82
1.67
1.7
2.8
1.66
21
34
1.59
7.3
12
1.59
5.5
8.6
1.57
0.4
0.5
1.35
44
48
1.09
0.4
0.4
1.08
4.6
4.9
1.07
3.0
3.2
1.06
101
105
1.04
6.4
6.6
1.02
650
563
0.87
8.6
6.9
0.81
198
146
0.74
21
15
0.72
198
118
0.60
345
200
0.58
7.1
3.9
0.55
1.9
1.0
0.53
4.8
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
97
2.6
0.53
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Hypoxanthine
Anthranilic acid
Spermidine
Met
Spermine
Carnosine
Thymidine
Inosine
Glutathione (GSH)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.D.; not detected.
N.A.; not available.
Table SIII-3. Concentration of fumarate in starved culture of R. palustris CGA009.
a
Incubation conditions
Fumarate concentration (µmol L-1)a
Dark 5day vialb-1
15.22
Dark 5day vial-2
12.06
Light 5day vial-1
N.D.
Light 5day vial-2
N.D.
Light 5day vial-3
N.D.
Fumarate in supernatant of starved culture in R. palustris was checked by HPLC
analysis.
b
Data are presented as the means of two (dark) or three (light) independent vials.
N.D.; not detected.
98
Table SIII-4. 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.
Acc. No.
rpa_rna52
rpa_rna51
rpa_rna57
rpa_rna58
rpa_rna50
rpa_rna59
rpa_rna3
rpa_rna34
rpa_rna48
rpa_rna47
rpa_rna28
rpa_rna41
rpa_rna33
rpa_rna2
rpa_rna6
rpa_rna21
rpa_rna10
rpa_rna9
rpa_rna60
rpa_rna5
rpa_rna30
rpa_rna23
rpa_rna7
rpa_rna12
rpa_rna14
rpa_rna36
rpa_rna4
rpa_rna43
rpa_rna40
rpa_rna11
rpa_rna49
rpa_rna8
rpa_rna35
rpa_rna26
rpa_rna15
rpa_rna37
rpa_rna29
rpa_rna42
rpa_rna39
rpa_rna17
rpa_rna20
rpa_rna24
rpa_rna19
rpa_rna16
rpa_rna46
rpa_rna56
rpa_rna18
rpa_rna31
The high expressed genes in the starved cells of R. palustris CGA009 in the light
definition
COG
r16S_1_16S rRNA 16S ribosomal RNA
RNA
r23S_1_23S rRNA 23S ribosomal RNA
RNA
rrnB_Ribosomal RNA
RNA
r23S_2_23S rRNA 23S ribosomal RNA
RNA
r5S_1_5S rRNA 5S ribosomal RNA
RNA
rnpB_unknown type
RNA
tRNA-Leu1_Transfer RNAs
RNA
tRNA-Ser1_Transfer RNAs
RNA
tRNA-Ala4_Transfer RNAs
RNA
tRNA-Ile2_Transfer RNAs
RNA
tRNA-Asp1_Transfer RNAs
RNA
tRNA-Ala2_Transfer RNAs
RNA
tRNA-Glu2_Transfer RNAs
RNA
tRNA-Thr1_Transfer RNAs
RNA
tRNA-Ala1_Transfer RNAs
RNA
tRNA-Gln2_Transfer RNAs
RNA
tRNA-Gln1_Transfer RNAs
RNA
tRNA-Val1_Transfer RNAs
RNA
ffs_unknown type
RNA
tRNA-Glu1_Transfer RNAs
RNA
tRNA-Asn1_Transfer RNAs
RNA
tRNA-Tyr1_Transfer RNAs
RNA
tRNA-Arg1_Transfer RNAs
RNA
tRNA-Met1_Transfer RNAs
RNA
tRNA-Met3_Transfer RNAs
RNA
tRNA-Val3_Transfer RNAs
RNA
tRNA-Thr2_Transfer RNAs
RNA
tRNA-Ser4_Transfer RNAs
RNA
tRNA-Ser3_Transfer RNAs
RNA
tRNA-Pro1_Transfer RNAs
RNA
tRNA-His1_Transfer RNAs
RNA
tRNA-Arg2_Transfer RNAs
RNA
tRNA-Ser2_Transfer RNAs
RNA
tRNA-Leu3_Transfer RNAs
RNA
tRNA-Lys1_Transfer RNAs
RNA
tRNA-Leu5_Transfer RNAs
RNA
tRNA-Lys2_Transfer RNAs
RNA
tRNA-Gly3_Transfer RNAs
RNA
tRNA-Gly2_Transfer RNAs
RNA
tRNA-Pro3_Transfer RNAs
RNA
tRNA-Thr3_Transfer RNAs
RNA
tRNA-Gly1_Transfer RNAs
RNA
tRNA-Arg3_Transfer RNAs
RNA
tRNA-Pro2_Transfer RNAs
RNA
tRNA-Phe1_Transfer RNAs
RNA
tmRNA_unknown type
RNA
tRNA-Val2_Transfer RNAs
RNA
tRNA-Cys1_Transfer RNAs
RNA
99
signal %
18.3666
15.4373
9.8257
5.7376
3.5223
2.5134
0.8611
0.5920
0.5629
0.5498
0.4538
0.4294
0.2907
0.2860
0.2802
0.2667
0.2220
0.2093
0.1644
0.1537
0.1386
0.1335
0.1149
0.1144
0.1123
0.1059
0.1001
0.0970
0.0933
0.0919
0.0912
0.0811
0.0779
0.0747
0.0546
0.0530
0.0453
0.0437
0.0426
0.0392
0.0369
0.0361
0.0324
0.0319
0.0286
0.0263
0.0233
0.0221
rpa_rna38
rpa_rna25
tRNA-Arg5_Transfer RNAs
tRNA-Trp1_Transfer RNAs
rpa1206
aldehyde dehydrogenase
rpa4394
isocitrate lyase
rpa4822
possible alcohol dehydrogenase
rpa1535
cytochrome c2
rpa4666
rpa0672
rpa4669
rpa1192
rpa4667
rpa1955
rpa3450
carbon-monoxide dehydrogenase small
subunit
4-hydroxybenzoyl-CoA reductase, third of
three subunits
putative racemase
cytochrome b6-F complex iron-sulfur
subunit
putative carbon-monoxide dehydrogenase
large subunit
glutathione dependent formaldehyde
dehydrogenase
putative malonic semialdehyde oxidative
decarboxylase
rpa0016
cytochrome-c oxidase fixP chain
rpa3392
possible nifU homolog
rpa1733
putative ferredoxin
rpa3215
putative nitroreductase
rpa1224
rpa3803
putative indolepyruvate ferredoxin
oxidoreductase, alpha subunit
carbon-monoxide dehydrogenase small
subunit
rpa2153
aldehyde dehydrogenase
rpa0059
L-carnitine dehydratase/bile acid-inducible
protein F
rpa2314
cytochrome c556
rpa1205
putative alcohol dehydrogenase
rpa4750
rpa1668
electron transfer flavoprotein beta chain,
(ETFSS)
Mg-protoporphyrin IX monomethyl ester
oxidative cyclase 66kD subunit
rpa0360
phosphoenolpyruvate carboxykinase
rpa0018
cytochrome-c oxidase fixO chain
rpa2458
putative cytochrome b561
rpa4410
glycerol-3-phosphate dehydrogenase
100
RNA
RNA
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
0.0196
0.0193
0.0665
0.0621
0.0508
0.0478
0.0469
0.0342
0.0318
0.0316
0.0279
0.0279
0.0266
0.0263
0.0259
0.0234
0.0214
0.0209
0.0201
0.0188
0.0184
0.0174
0.0173
0.0163
0.0160
0.0156
0.0138
0.0136
0.0133
rpa3802
carbon monoxide dehydrogenase medium
subunit
rpa1991
conserved hypothetical protein
rpa3834
NADP-dependent isocitrate dehydrogenase
rpa1532
geranylgeranyl reductase
prpa2
partition protein
rpa4077
ATPase, ParA type
rpa1622
conserved unknown protein
rpa3033
possible acetylornitine deacetylase
rpa0870
putative ornithine decarboxylase
rpa4020
rpa3810
rpa1789
rpa2966
rpa3093
rpa3725
possible branched-chain amino acid
transport system permease protein
putative periplasmic binding protein of
ABC transporter
putative branched-chain amino acid
transport system substrate-binding protein
nitrogen regulatory protein P-II
possible urea/short-chain binding protein
of ABC transporter
possible leucine/isoleucine/valine-binding
protein precursor
rpa2046
2-isopropylmalate synthase
rpa2446
putative aminotransferase
rpa1798
putative periplasmic binding protein for
ABC transporter for branched chain amino
acids
rpa0230
aspartate-semialdehyde dehydrogenase
rpa0668
putative ABC transporter subunit,
substrate-binding component
rpa2503
possible aminotransferase
rpa4807
rpa3297
rpa4331
rpa0557
rpa1664
rpa3724
possible branched-chain amino acid
transport system substrate-binding protein
possible branched-chain amino acid
transport system substrate-binding protein
aspartate aminotransferase A
cysteine synthase, cytosolic
O-acetylserine(thiol)lyase
Glyoxalase/Bleomycin resistance
protein/dioxygenase domain
periplasmic binding protein
101
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Cell Division and Chromosome
Partitioning
Cell Division and Chromosome
Partitioning
Cell Division and Chromosome
Partitioning
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
0.0121
0.0119
0.0118
0.0114
0.0606
0.0249
0.0139
1.0201
0.9082
0.4570
0.1635
0.1061
0.1030
0.0920
0.0807
0.0709
0.0686
0.0578
0.0549
0.0545
0.0472
0.0416
0.0409
0.0403
0.0391
0.0387
0.0385
rpa4019
rpa3669
rpa4772
rpa1651
rpa0027
rpa1741
rpa2763
rpa2193
rpa3719
rpa2491
putative branched-chain amino acid ABC
transporter system substrate-binding
protein
putative urea short-chain amide or
branched-chain amino acid uptake ABC
transporter periplasmic solute-binding
protein precursor
ornithine carbamoyltransferase
possible leucine/isoleucine/valine-binding
protein precursor
2-dehydro-3-deoxyphosphoheptonate
aldolase
possible branched-chain amino acid
transport system substrate-binding protein
putative O-acetylhomoserine sulfhydrylase
putative ABC transporter, perplasmic
binding protein, branched chain amino
acids
putative high-affinity branched-chain
amino acid transport system ATP-binding
protein
N-acetylglutamate semialdehyde
dehydrogenase
rpa2724
glycine hydroxymethyltransferase
rpa4813
possible branched chain amino acid
periplasmic binding protein of ABC
transporter
rpa4209
glutamine synthetase II
rpa0235
3-isopropylmalate dehydratase small
subunit
rpa4179
conserved unknown protein
rpa1283
rpa4034
homoserine/homoserine lactone/threonine
efflux protein
ABC transporter, periplasmic branched
chain amino acid binding protein
leucine aminopeptidase
rpa4773
putative acetylornithine aminotransferase
rpa0240
3-isopropylmalate dehydratase
rpa3429
serine acetyltransferase
rpa1415
possible branched-chain amino acid
transport system substrate-binding protein
rpa2166
conserved hypothetical protein
rpa1984
0.0382
Amino Acid Transport and
Metabolism
0.0353
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
putative branched-chain amino acid ABC
transport system ATP-binding protein
2-dehydro-3-deoxyphosphoheptonate
aldolase
102
0.0351
0.0347
0.0346
0.0341
0.0242
Amino Acid Transport and
Metabolism
0.0241
Amino Acid Transport and
Metabolism
0.0230
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
rpa3060
rpa4041
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
0.0228
0.0228
0.0211
0.0210
0.0194
0.0189
0.0182
0.0182
0.0170
0.0162
0.0161
0.0140
0.0136
0.0135
0.0123
0.0121
rpa0985
rpa1655
putative branched-chain amino acid
transport system substrate- binding protein
possible urea/short-chain binding protein
of ABC transporter
rpa2967
glutamine synthetase I
rpa4071
carbamoyl-phosphate synthase large
subunit
rpa2200
inosine monophosphate dehydrogenase
rpa3821
phosphoribosylaminoimidazole-succinocar
boxamide synthase
rpa1890
hypothetical protein
rpa1069
possible dihydroorotase
rpa4601
conserved hypothetical protein
rpa1276
rpa2047
rpa3470
rpa0944
carbamoyl-phosphate synthase small
subunit
TrapT family, dctP subunit,
C4-dicarboxylate periplasmic binding
protein
putative sugar uptake ABC transporter
periplasmic solute-binding protein
precursor
glyceraldehyde-3-phosphate
dehydrogenase(GAPDH)
rpa1981
ribose 5-phosphate isomerase
rpa1559
ribulose-bisphosphate carboxylase large
chain
rpa0851
possible MFS transporter
rpa0940
fructose-bisphosphate aldolase
rpa3039
possible polysaccharide deacetylase
rpa2782
TrapT family, dctP subunit,
C4-dicarboxylate periplasmic binding
protein
rpa3825
conserved hypothetical protein
rpa3471
ABC transporter, ATP-binding protein
rpa4432
NHL repeat
rpa2049
TrapT family, dctM subunit,
C4-dicarboxylate transport
rpa0355
putative pts system permease (IIAMan)
rpa1055
rpa0300
quinolinate synthetase A
putative dephospho-CoA kinase CoaE
putative branched-chain amino acid
aminotransferase
geranylgeranyl pyrophosphate synthase
delta-aminolevulinic acid dehydratase
rpa4370
rpa1519
rpa2712
103
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
0.0121
0.0120
0.0113
0.0285
0.0170
0.0154
0.0149
0.0136
0.0122
0.0116
Carbohydrate Transport and
Metabolism
0.2409
Carbohydrate Transport and
Metabolism
0.0394
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
0.0341
0.0328
0.0290
0.0247
0.0172
0.0164
0.0159
0.0156
0.0149
0.0146
0.0135
0.0114
0.4340
0.1000
Coenzyme Metabolism
0.0829
Coenzyme Metabolism
Coenzyme Metabolism
0.0640
0.0622
rpa0165
rpa1054
rpa0854
rpa0930
rpa0875
rpa2671
rpa2035
rpa1666
rpa4309
rpa2089
rpa4719
rpa1546
rpa1257
rpa1179
rpa1040
rpa1554
rpa2031
rpa3391
rpa2587
rpa4002
rpa3075
rpa3441
rpa3717
rpa4346
rpa0211
rpa3446
rpa4748
rpa1729
rpa2918
rpa4155
rpa1603
rpa3203
rpa1094
rpa2007
rpa0071
rpa0661
rpa0513
rpa3289
rpa2006
rpa0818
possible nicotinate-nucleotide
adneylyltransferase
putative L-aspartate oxidase
5-aminolevulinic acid synthase (ALAS)
possible 3-octaprenyl-4-hydroxybenzoate
carboxy-lyase
ferrochelatase
putative FAD-dependent monoxygenase
ketol-acid reductoisomerase
putative coproporphyrinogen oxidase III
phosphoserine aminotransferase,
precorrin 2 methylase
molybdo-pterin binding protein
Mg-protoporphyrin IX methyl transferase
possible cobalamin (5`-phosphate)
synthase
beta-alanine-pyruvate transaminase
octaprenyl-diphosphate synthase
5-aminolevulinic acid synthase (ALAS)
acetolactate synthase (large subunit)
possible GTP cyclohydrolase I
lipoic acid synthetase
putative acyl-CoA dehydrogenase
putative malonyl CoA-acyl carrier protein
transacylase
enoyl-CoA hydratase/isomerase family
protein
enoyl-CoA hydratase
putative acetyl-CoA acyltransferase
acetyl-CoA synthetase
3-hydroxyisobutyrate dehydrogenase
3-hydroxybutyryl-CoA dehydrogenase
Fatty acid desaturase family
undecaprenyl pyrophosphate synthetase
3-oxoadipate CoA-transferase subunit B
putative fatty acid desaturase
propionyl-CoA carboxylase beta chain
glutaryl-CoA dehydrogenase
possible phosphatidylserine synthase
acetyl-CoA carboxylase
carboxyltransferase beta subunit
benzoate-CoA ligase
putative acetyl-CoA acetyltransferase
acyl-CoA dehydrogenase
putative phosphatidylserine decarboxylase
probable 3-hydroxyacyl-CoA
dehydrogenase
rpa0159
ribosomal protein L27
rpa3225
50S ribosomal protein L17
rpa0160
possible acetyltransferases.
rpa3270
50S ribosomal protein L10
rpa0433
ribosomal protein S15
Coenzyme Metabolism
0.0490
Coenzyme Metabolism
Coenzyme Metabolism
0.0472
0.0300
Coenzyme Metabolism
0.0290
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
0.0288
0.0277
0.0228
0.0205
0.0190
0.0189
0.0179
0.0166
Coenzyme Metabolism
0.0162
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
Lipid Metabolism
0.0156
0.0154
0.0135
0.0132
0.0127
0.0116
0.1004
Lipid Metabolism
0.0462
Lipid Metabolism
0.0318
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0301
0.0274
0.0267
0.0219
0.0213
0.0186
0.0184
0.0162
0.0161
0.0159
0.0151
0.0144
Lipid Metabolism
0.0143
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0128
0.0125
0.0123
0.0118
Lipid Metabolism
0.0115
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
104
0.1592
0.1495
0.0910
0.0869
0.0694
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
rpa3227
30S ribosomal protein S11
rpa3228
30S ribosomal protein S13
rpa0039
50S ribosomal protein L35
rpa1589
30S ribosomal protein S4
rpa3252
elongation factor Tu
rpa0051
putative sigma-54 modulation protein
rpa0040
translation initiation factor IF-3
rpa0038
ribosomal protein L20
rpa0493
50S ribosomal protein L28
rpa3269
50S ribosomal protein L7/L12
rpa0526
50S ribosomal protein L32
rpa0918
possible 50S ribosomal protein L31
rpa4328
elongation factor G, EF-G
rpa0867
Endoribonuclease L-PSP
rpa4176
ribosomal protein S21
rpa3255
30S ribosomal protein S12
rpa4197
50S ribosomal protein L36
rpa3129
50S ribosomal protein L33
rpa0158
putative ribosomal protein L21
rpa2768
ribosomal protein S9
rpa2922
30S ribosomal protein S2
rpa4356
putative 50S ribosomal protein L25
rpa3111
Glu-tRNA(Gln) amidotransferase subunit
C
rpa4836
30S ribosomal protein S20
rpa2651
Regulator of chromosome condensation,
RCC1:Endoribonuclease L-PSP
rpa3137
Endoribonuclease L-PSP
rpa0621
putative
N-formylmethionylaminoacyl-tRNA
deformylase
Translation, Ribosomal Structure
and Biogenesis
0.0134
rpa3233
ribosomal protein S5
Translation, Ribosomal Structure
and Biogenesis
0.0132
105
0.0653
0.0503
0.0458
0.0418
0.0397
0.0381
0.0354
0.0334
0.0319
0.0277
0.0273
0.0267
0.0257
0.0243
0.0242
0.0235
0.0221
0.0178
0.0177
0.0175
0.0157
0.0154
0.0152
0.0151
0.0149
0.0134
rpa3272
50S ribosomal protein L1
rpa0241
50s ribosomal protein L19
rpa2777
methionyl-tRNA synthetase
rpa3672
rpa3399
rpa1173
rpa2490
rpa4225
rpa0488
rpa4072
rpa2692
rpa0913
rpa2743
cold shock protein
cold shock protein
possible cold shock protein
conserved hypothetical protein
putative RNA polymerase sigma factor
possible CarD-like transcriptional regulator
transcriptional elongation factor greA
RNA polymerase omega subunit
DUF179
possible transcriptional regulator, MarR
family
DNA-directed RNA polymerase alpha
subunit
stress response sigma factor (sigma37,
sigma32 family)
putative RNA polymerase sigma-E factor
putative transcriptional regulator
Transcriptional Regulator, AraC family
transcriptional regulator
possible transcriptional regulator
putative nucleoside diphosphate kinase
regulator
bacterial regulatory protein, MerR family
rpa0207
unknown protein
rpa4277
conserved hypothetical protein
rpa2953
possible DNA-binding protein hu-alpha
(NS2) (HU-2)
rpa3851
recombination protein recA
rpa2742
integration host factor alpha subunit
rpa0782
conserved unknown protein
rpa0066
integration host factor beta chain
rpa2814
single-strand DNA-binding protein
prpa5
resolvase
rpa3020
RND multidrug efflux membrane fusion
protein MexE precursor
rpa0857
possible outer membrane protein
rpa3371
putative outer membrane protein
rpa0169
putative periplasmic carboxyl-terminal
processing protease
rpa2686
dihydrodipicolinate synthase
rpa2715
rpa3226
rpa0367
rpa4792
rpa3718
rpa2140
rpa3021
rpa0443
rpa0866
106
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
0.0127
0.0126
0.0115
0.1721
0.0622
0.0567
0.0514
0.0433
0.0331
0.0330
0.0276
0.0256
Transcription
0.0250
Transcription
0.0248
Transcription
0.0247
Transcription
Transcription
Transcription
Transcription
Transcription
0.0204
0.0197
0.0179
0.0167
0.0154
Transcription
0.0153
Transcription
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
0.0150
0.0861
0.0663
0.0534
0.0256
0.0252
0.0147
0.0139
0.0123
0.0117
0.1457
0.0884
0.0472
0.0449
0.0411
rpa1123
omp16 protein
rpa0222
Beta-Ig-H3/Fasciclin domain
rpa2772
rare lipoprotein A
rpa1917
LipA a lipoprotein
rpa1172
possible outer membrane protein
rpa2837
Peptidoglycan-binding LysM:Peptidase
M23/M37
rpa3536
putative penicillin-binding protein
rpa4502
putative outer membrane protein
rpa4271
penicillin tolerance protein (lytB) , control
of stringent response
rpa1879
Choloylglycine hydrolase
rpa4678
rpa1807
possible outer membrane protein OprF
(AF117972)
possible heavy metal efflux pump, HlyD
family secretion protein
rpa0805
possible outer membrane protein
rpa3929
rpa2033
conserved unknown protein
possible AtsE
rpa0889
small heat shock protein
rpa0453
possible NifU-like domain (residues
119-187)
rpa2895
possible small heat shock protein
rpa2959
ATP-dependent protease Lon
rpa1929
htrA-like serine protease
rpa0787
putative heat shock protein (htpX)
rpa0333
heat shock protein DnaK (70)
rpa0054
putative small heat shock protein
rpa1126
rpa2960
metalloprotease (cell division protein)
FtsH
ATP-dependent Clp protease ATP binding
subunit ClpX
rpa3812
putative holocytochrome c synthase
rpa2165
chaperonin GroES2, cpn10
rpa3491
putative protease subunit hflK
rpa2443
probable antioxidant protein
107
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Motility and Secretion
Cell Motility and Secretion
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
0.0244
0.0242
0.0205
0.0193
0.0171
0.0151
0.0148
0.0142
0.0138
0.0126
0.0125
0.0118
0.0118
0.0587
0.0167
0.2799
0.1097
0.1041
0.1007
0.0978
0.0965
0.0909
0.0769
0.0755
0.0738
0.0459
0.0445
0.0436
0.0404
rpa4579
possible serine protease, htrA-like
rpa3147
endopeptidase Clp: ATP-binding chain A
rpa4268
peroxiredoxin-like protein
rpa0019
rpa0966
rpa0017
cytochrome-c oxidase fixN chain, heme
and copper binding subunit
putative membrane-bound hydrogenase
component hupE
cytochrome oxidase subunit, small
membrane protein
rpa4487
DSBA oxidoreductase:Tat pathway signal
rpa1140
chaperonin GroEL1, cpn60
rpa0331
possible heat shock protein (HSP-70
COFACTOR), grpE
rpa1606
conserved unknown protein
rpa0373
thioredoxin
rpa3159
probable glutathione S-transferase
rpa2461
Protein of unknown function UPF0075
rpa1320
conserved hypothetical protein
rpa4069
DUF25
rpa1576
putative glutathione S-transferase
rpa3799
DUF182
rpa3490
putative hflC protein
rpa4194
osmotically inducible protein OsmC
rpa0073
thioredoxin
rpa0452
Glycoprotease (M22) metalloprotease
rpa0598
putative glutaredoxin
rpa3488
probable serine protease
rpa2720
possible glutathione-S-transferase
rpa2164
chaperonin GroEL2, cpn60
rpa2442
putative outer membrane protein
rpa4075
thioredoxin reductase
rpa1022
possible outer membrane protein
108
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
0.0397
0.0326
0.0318
0.0297
0.0293
0.0265
0.0241
0.0227
0.0224
0.0220
0.0198
0.0188
0.0183
0.0183
0.0176
0.0172
0.0170
0.0162
0.0154
0.0154
0.0143
0.0140
0.0138
0.0135
0.0130
0.0121
0.0121
0.0120
rpa3627
putative glutathione peroxidase
rpa3937
putative transcriptional regulator
rpa0429
catalase/peroxidase
rpa1693
superoxide dismutase
rpa3600
bacterioferritin
rpa0225
putative superoxide dismutase (Cu/Zn)
rpa1522
rpa3002
bacteriochlorophyllide reductase subunit
BchX
potassium-transporting atpase c chain,
KdpC
rpa2269
conserved unknown protein
rpa0450
ferric uptake regulation protein
rpa4757
possible outer membrane receptor for iron
transport
rpa0424
iron response regulator protein
rpa2043
putative ABC transporter, periplasmic
substrate-binding protein
rpa4636
FeoA family
rpa4717
putative molybdate transport system
substrate-binding protein
rpa4620
nitrogenase iron protein, nifH
rpa2121
conserved unknown protein
rpa1274
rpa0013
possible Dps protein family
starvation-inducible DNA-binding protein
putative cation (heavy metal) transporting
ATPase
rpa1975
possible Trap-T transport system, dctP
subunit
rpa4443
acyl-CoA synthetase
rpa4267
long-chain-fatty-acid-CoA-ligase
rpa3299
putative long-chain-fatty-acid CoA ligase
rpa1763
putative long-chain-fatty-acid CoA ligase
rpa1207
putative transcriptional regulator
109
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
0.0117
0.0117
0.0753
0.0431
0.0347
0.0322
0.0310
0.0301
0.0277
0.0252
0.0193
0.0171
0.0152
0.0152
0.0149
0.0132
0.0123
0.0122
0.0114
0.1042
0.0526
0.0276
0.0259
0.0255
0.0243
rpa3868
putatively similar to frnE protein
rpa3767
phenylacetic acid degradation protein paaB
rpa2714
putative long-chain-fatty-acid--CoA ligase
rpa3073
constitutive acyl carrier protein
rpa0743
putative acid-CoA ligase
rpa3072
3-oxoacyl-acyl carrier protein synthase II
rpa3961
putative periplasmic binding protein for
ABC transporter
rpa0865
homospermidine synthase
rpa2780
possible AMP-binding enzyme
rpa0198
rpa0869
rpa0460
rpa2869
conserved hypothetical protein
GCN5-related N-acetyltransferase
putative esterase
possible flavin-dependent oxidoreductase
possible TrapT family, dctP subunit,
C4-dicarboxylate periplasmic binding
protein
putative 3-oxoacyl-[ACP] reductase
conserved unknown protein
putative molybdate transport system
regulatory protein
Flp/Fap pilin component
probable alcohol dehydrogenase
putative 3-ketoacyl-CoA reductase
putative 3-oxoacyl-acyl carrier protein
reductase
possible penicillin binding protein
putative NAD-dependent alcohol
dehydrogenase
4-hydroxyphenylpyruvate dioxygenase
PAP/25A core domain:DNA polymerase,
beta-like region
quinone oxidoreductase
possible 2-nitropropane dioxygenase
probable hydrolase
possible 3-hydroxyacyl-CoA
dehydrogenase type II
conserved hypothetical protein
putative hemmolysin III, HlyIII family
rpa3458
rpa0895
rpa3501
rpa4718
rpa3675
rpa3655
rpa2417
rpa0896
rpa3284
rpa3067
rpa0005
rpa0276
rpa2201
rpa1941
rpa0568
rpa1705
rpa2823
rpa1585
110
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0226
0.0209
0.0205
0.0182
0.0154
0.0146
0.0142
0.0138
0.0118
0.6308
0.2524
0.0958
0.0580
General Function Prediction Only
0.0568
General Function Prediction Only
General Function Prediction Only
0.0430
0.0416
General Function Prediction Only
0.0387
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0349
0.0324
0.0314
General Function Prediction Only
0.0286
General Function Prediction Only
0.0281
General Function Prediction Only
0.0277
General Function Prediction Only
0.0264
General Function Prediction Only
0.0241
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0235
0.0233
0.0232
General Function Prediction Only
0.0231
General Function Prediction Only
General Function Prediction Only
0.0228
0.0225
rpa2849
rpa2667
rpa4337
rpa1521
rpa2848
rpa4786
rpa0642
rpa0709
rpa2488
rpa2862
rpa3223
rpa0532
rpa0534
rpa2809
rpa1940
rpa0400
rpa3287
rpa2105
rpa4510
rpa2811
rpa2282
rpa3377
rpa2934
rpa1594
rpa1185
rpa3504
rpa1277
rpa3881
rpa1146
rpa1623
rpa4272
rpa1133
rpa0530
rpa3565
rpa1330
rpa0053
rpa1747
rpa3947
rpa1659
rpa2711
rpa3511
rpa0089
rpa2470
rpa4357
rpa4138
rpa3421
rpa3768
rpa0403
putative sec-independent protein
translocase protein tatA/E
conserved unknown protein
putative phosphoglycolate phosphatase
2-desacetyl-2-hydroxyethyl
bacteriochlorophyllide a dehydrogenase
possible sec-independent protein secretion
pathway component TatB
putative beta-ketoacyl reductase
conserved hypothetical protein
conserved unknown protein
conserved unknown protein
possible exopolysaccharide regulatory
protein exoR
putative alginate lyase
beta-ketothiolase, acetoacetyl-CoA
reductase
conserved hypothetical protein
Beta-lactamase
putative quinone oxidoreductase
possible alcohol dehydrogenases and
quinone oxidoreductases.
putative 3-oxoacyl-acyl carrier protein
reductase
non-heme chloroperoxidase
conserved unknown protein
possible mazG
conserved hypothetical protein
3-oxoacyl-acyl carrier protein reductase
conserved unknown protein
putative tryptophan synthase beta chain
putative methanol dehydrogenase regulator
Thioesterase superfamily
possible beta-ketoadipate enol-lactone
hydrolase
conserved unknown protein
conserved unknown protein
conserved unknown protein
conserved unknown protein
unknown protein
conserved unknown protein
ErfK/YbiS/YcfS/YnhG
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
ErfK/YbiS/YcfS/YnhG
conserved unknown protein
Protein of unknown function,
HesB/YadR/YfhF
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
phenylacetic acid degradation protein paaA
conserved hypothetical protein
111
General Function Prediction Only
0.0218
General Function Prediction Only
General Function Prediction Only
0.0216
0.0216
General Function Prediction Only
0.0208
General Function Prediction Only
0.0207
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0201
0.0199
0.0192
0.0188
General Function Prediction Only
0.0178
General Function Prediction Only
0.0169
General Function Prediction Only
0.0167
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0161
0.0157
0.0156
General Function Prediction Only
0.0154
General Function Prediction Only
0.0153
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0153
0.0150
0.0135
0.0131
0.0129
0.0125
0.0122
0.0117
0.0117
General Function Prediction Only
0.0114
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.1845
0.0970
0.0888
0.0828
0.0582
0.0490
0.0489
0.0477
0.0440
0.0434
0.0433
0.0430
0.0427
0.0413
0.0381
Function Unknown
0.0376
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0341
0.0298
0.0283
0.0281
0.0265
rpa2288
rpa1092
rpa3568
rpa3148
rpa4467
rpa0477
rpa3037
rpa3611
rpa0847
rpa3822
rpa0303
rpa3432
rpa1278
rpa0060
rpa4137
rpa2856
rpa3099
rpa4284
rpa1007
rpa3659
rpa3770
rpa0166
rpa1690
rpa0152
rpa3081
rpa4442
rpa0917
rpa3109
rpa2691
rpa0258
rpa1617
rpa2732
rpa3499
rpa1119
rpa3178
rpa4250
rpa0571
rpa1515
rpa3054
rpa0283
rpa1632
rpa0852
rpa2903
rpa4234
rpa2560
rpa4245
rpa4249
rpa2839
rpa2111
rpa0249
Streptomyces cyclase/dehydrase
Carboxymuconolactone decarboxylase
conserved unknown protein
DUF174
putative sulfur oxidation protein soxY
conserved hypothetical protein
conserved unknown protein
ErfK/YbiS/YcfS/YnhG
FoF1 ATP synthase, subunit I
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
GatB/Yqey
conserved unknown protein
conserved unknown protein
Protein of unknown function,
HesB/YadR/YfhF
conserved hypothetical protein
YceI like family
possible 2,3-dihydroxyphenylpropionate
1,2-dioxygenase
DUF176
conserved unknown protein
Iojap-related protein
conserved unknown protein
Protein of unknown function, UPF0066
unknown protein
ErfK/YbiS/YcfS/YnhG
Transcriptional Regulator, AraC Family
conserved hypothetical protein
DUF88
unknown protein
ErfK/YbiS/YcfS/YnhG
conserved hypothetical protein
conserved unknown protein
putative TolA
hypothetical protein
nitrogen fixation regulatory protein fixK2
two-component, response regulator
conserved unknown protein
conserved unknown protein
putative two-component response regulator
two-component transcriptional regulator
probable two-component system, response
regulator
SOS response regulator lexA protein
anaerobic aromatic degradation regulator
aadR
ABC transporter, periplasmic amino acid
binding protein aapJ-1
conserved unknown protein
putative two-component response regulator
Response regulator receiver
probable transcriptional regulator
hypothetical protein
112
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0264
0.0251
0.0247
0.0238
0.0236
0.0226
0.0210
0.0207
0.0206
0.0198
0.0196
0.0196
0.0186
0.0185
0.0185
Function Unknown
0.0180
Function Unknown
Function Unknown
0.0172
0.0172
Function Unknown
0.0171
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
0.0159
0.0154
0.0153
0.0151
0.0143
0.0138
0.0138
0.0137
0.0129
0.0128
0.0125
0.0124
0.0123
0.0121
0.0119
0.0113
0.8829
0.1790
0.0837
0.0712
0.0595
0.0408
Signal Transduction Mechanisms
0.0336
Signal Transduction Mechanisms
0.0320
Signal Transduction Mechanisms
0.0235
Signal Transduction Mechanisms
0.0221
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
0.0205
0.0198
0.0185
0.0173
0.0170
rpa4830
rpa3940
rpa2050
rpa1491
rpa1492
rpa4292
rpa4291
rpa1665
rpa3601
rpa3013
rpa1263
rpa1637
rpa0868
rpa3158
rpa1474
rpa2654
rpa0259
rpa1526
rpa2023
rpa4128
rpa4393
rpa2444
rpa3878
rpa2653
rpa1114
rpa3875
rpa0538
rpa4375
rpa4704
rpa1495
rpa2801
rpa3602
rpa4239
rpa1525
rpa3373
rpa2134
rpa4525
rpa3180
rpa1309
rpa1577
rpa3103
Response regulator
receiver:Metal-dependent
phosphohydrolase, HD region
Universal stress protein (Usp)
conserved hypothetical protein
light harvesting protein B-800-850, beta
chain E (antenna pigment protein, beta
chain E) (LH II-E beta)
light harvesting protein B-800-850, alpha
chain E (antenna pigment protein, alpha
chain E) (LH II-E alpha)
light harvesting protein B-800-850, alpha
chain B (antenna pigment protein, alpha
chain B) (LH II-B alpha)
light harvesting protein B-800-850, beta
chain B (antenna pigment protein, beta
chain B)
hypothetical protein
unknown protein
light harvesting protein B-800-850, beta
chain D (antenna pigment protein, beta
chain D) (LH II-D beta)
putative II.1 protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
light harvesting protein B-800-850, beta
chain A (antenna pigment protein, beta
chain A) (LH II-A beta)
Histidine kinase, HAMP region
light-harvesting complex 1 alpha chain
conserved hypothetical protein
conserved hypothetical protein
unknown protein
unknown protein
conserved unknown protein
light harvesting protein B-800-850, alpha
chain A (antenna pigment protein, alpha
chain A) (LH II-A alpha)
conserved unknown protein
conserved unknown protein
putative Omp2b porin
unknown protein
conserved hypothetical protein
unknown protein
Collagen triple helix repeat
unknown protein
conserved unknown protein
light-harvesting complex 1 beta chain
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
possible transposase
hypothetical protein
hypothetical protein
113
Signal Transduction Mechanisms
0.0139
Signal Transduction Mechanisms
not in COGs
0.0128
1.0230
not in COGs
0.5562
not in COGs
0.4144
not in COGs
0.3843
not in COGs
0.3465
not in COGs
not in COGs
0.2944
0.1916
not in COGs
0.1891
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.1866
0.1517
0.1514
0.1491
0.1300
not in COGs
0.1192
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.1150
0.1043
0.1015
0.0963
0.0944
0.0941
0.0941
not in COGs
0.0920
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0890
0.0872
0.0822
0.0818
0.0792
0.0722
0.0713
0.0707
0.0692
0.0691
0.0628
0.0624
0.0580
0.0566
0.0556
0.0532
0.0524
rpa4330
rpa2199
rpa2717
rpa2991
rpa0705
rpa3912
rpa3510
rpa3197
rpa3012
rpa4349
rpa1070
rpa2656
rpa4683
rpa0710
rpa1006
rpa1152
rpa1549
rpa0864
rpa4183
rpa2419
rpa0768
rpa0543
rpa4127
rpa2716
rpa0091
rpa0925
rpa1091
rpa1653
rpa4805
rpa4201
rpa3745
rpa4466
rpa4210
rpa1528
rpa1645
rpa1014
rpa1717
rpa4224
rpa2579
rpa4804
rpa2803
rpa0546
rpa3186
rpa1066
rpa1243
rpa2825
rpa4720
rpa2157
rpa2502
rpa1634
rpa3975
rpa4760
conserved unknown protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved unknown protein
hypothetical protein
light harvesting protein B-800-850, alpha
chain D (antenna pigment protein, alpha
chain D) (LH II-D alpha)
conserved unknown protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
possible protocatechuate 4,5-dioxygenase
small subunit (AB035121)
hypothetical protein
possible photosynthetic complex assembly
protein
hypothetical protein
hypothetical protein
possible outer membrane protein, possible
porin
possible transposase
unknown protein
conserved hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved unknown protein
conserved hypothetical protein
hypothetical protein
unknown protein
putative sulfur oxidation protein soxZ
hypothetical protein
photosynthetic reaction center M protein
unknown protein
conserved hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved unknown protein
conserved hypothetical protein
conserved hypothetical protein
unknown protein
conserved unknown protein
unknown protein
unknown protein
114
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0494
0.0485
0.0457
0.0451
0.0443
0.0439
0.0436
0.0411
not in COGs
0.0409
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0388
0.0380
0.0374
0.0372
0.0367
not in COGs
0.0365
not in COGs
0.0362
not in COGs
0.0355
not in COGs
not in COGs
0.0344
0.0333
not in COGs
0.0323
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0319
0.0313
0.0311
0.0309
0.0309
0.0302
0.0299
0.0295
0.0279
0.0276
0.0273
0.0272
0.0271
0.0269
0.0269
0.0263
0.0262
0.0259
0.0256
0.0255
0.0254
0.0252
0.0238
0.0236
0.0235
0.0231
0.0229
0.0229
0.0224
0.0223
0.0220
0.0219
rpa0228
rpa0871
rpa0260
rpa3328
rpa0238
rpa4068
rpa2652
rpa4329
rpa0849
rpa2894
rpa4293
rpa4232
rpa4347
rpa2439
rpa3378
rpa1211
rpa2516
rpa4222
rpa3024
rpa1527
rpa0608
rpa4574
rpa2547
rpa1431
rpa0366
rpa3456
rpa4079
rpa3083
rpa0926
rpa3935
rpa0090
rpa4154
rpa0801
rpa0634
rpa4230
rpa3744
rpa1134
rpa2537
rpa2600
rpa3334
rpa0845
rpa1258
rpa2747
rpa1142
rpa0088
rpa3874
rpa3292
rpa3110
rpa1620
rpa3035
rpa3860
rpa0885
rpa1111
unknown protein
conserved hypothetical protein
possible photosynthesis gene regulator,
AppA/PpaA family
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
conserved unknown protein
conserved hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
unknown protein
photosynthetic reaction center L subunit
conserved unknown protein
hypothetical protein
hypothetical protein
putative NAD+ ADP-ribosyltransferase
unknown protein
possible transcriptional regulator
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical protein
possible ribosomal protein L34
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
possible NADH dehydrogenase
(ubiquinone) Fe-S protein 4 (18kD)
(NADH-coenzyme Q reductase)
hypothetical protein
hypothetical protein
probable ATP synthase subunit C
TRANSMEMBRANE protein
hypothetical protein
hypothetical protein
unknown protein
unknown protein
hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
115
not in COGs
not in COGs
0.0213
0.0211
not in COGs
0.0201
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0198
0.0197
0.0196
0.0196
0.0191
0.0191
0.0190
0.0189
0.0186
0.0182
0.0181
0.0180
0.0180
0.0179
0.0179
0.0175
0.0175
0.0173
0.0173
0.0171
0.0170
0.0169
0.0167
0.0165
0.0164
0.0164
0.0163
0.0162
0.0160
0.0158
0.0157
0.0152
0.0149
0.0148
not in COGs
0.0146
not in COGs
not in COGs
0.0143
0.0143
not in COGs
0.0140
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0139
0.0139
0.0138
0.0133
0.0132
0.0130
0.0130
0.0128
0.0127
0.0127
0.0127
0.0126
rpa2245
rpa2603
rpa4534
rpa2171
rpa2969
rpa0200
rpa3773
rpa0117
rpa3787
rpa2853
rpa3428
rpa4003
rpa3423
rpa1223
hypothetical protein
conserved hypothetical protein
hypothetical protein
unknown protein
unknown protein
conserved hypothetical protein
conserved unknown protein
dTDP-D-glucose 4,6-dehydratase
hypothetical protein
Proline-rich region
conserved unknown protein
conserved hypothetical protein
conserved unknown protein
hypothetical protein
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
116
0.0125
0.0124
0.0122
0.0119
0.0119
0.0118
0.0118
0.0118
0.0117
0.0117
0.0114
0.0114
0.0114
0.0114
The high expressed genes in the starved cells of R. palustris CGA009 in the dark
Acc. No.
definition
COG
rpa_rna52 r16S_1_16S rRNA 16S ribosomal RNA
RNA
rpa_rna51 r23S_1_23S rRNA 23S ribosomal RNA
RNA
rpa_rna50 r5S_1_5S rRNA 5S ribosomal RNA
RNA
rpa_rna57 rrnB_Ribosomal RNA
RNA
rpa_rna58 r23S_2_23S rRNA 23S ribosomal RNA
RNA
rpa_rna59 rnpB_unknown type
RNA
rpa_rna33 tRNA-Glu2_Transfer RNAs
RNA
rpa_rna34 tRNA-Ser1_Transfer RNAs
RNA
rpa_rna3
tRNA-Leu1_Transfer RNAs
RNA
rpa_rna7
tRNA-Arg1_Transfer RNAs
RNA
rpa_rna28 tRNA-Asp1_Transfer RNAs
RNA
rpa_rna23 tRNA-Tyr1_Transfer RNAs
RNA
rpa_rna9
tRNA-Val1_Transfer RNAs
RNA
rpa_rna47 tRNA-Ile2_Transfer RNAs
RNA
rpa_rna41 tRNA-Ala2_Transfer RNAs
RNA
rpa_rna6
tRNA-Ala1_Transfer RNAs
RNA
rpa_rna10 tRNA-Gln1_Transfer RNAs
RNA
rpa_rna12 tRNA-Met1_Transfer RNAs
RNA
rpa_rna43 tRNA-Ser4_Transfer RNAs
RNA
rpa_rna48 tRNA-Ala4_Transfer RNAs
RNA
rpa_rna2
tRNA-Thr1_Transfer RNAs
RNA
rpa_rna60 ffs_unknown type
RNA
rpa_rna29 tRNA-Lys2_Transfer RNAs
RNA
rpa_rna21 tRNA-Gln2_Transfer RNAs
RNA
rpa_rna8
tRNA-Arg2_Transfer RNAs
RNA
rpa_rna40 tRNA-Ser3_Transfer RNAs
RNA
rpa_rna35 tRNA-Ser2_Transfer RNAs
RNA
rpa_rna49 tRNA-His1_Transfer RNAs
RNA
rpa_rna1
tRNA-Undet1_Transfer RNAs
RNA
rpa_rna36 tRNA-Val3_Transfer RNAs
RNA
rpa_rna30 tRNA-Asn1_Transfer RNAs
RNA
rpa_rna5
tRNA-Glu1_Transfer RNAs
RNA
rpa_rna37 tRNA-Leu5_Transfer RNAs
RNA
rpa_rna19 tRNA-Arg3_Transfer RNAs
RNA
rpa_rna26 tRNA-Leu3_Transfer RNAs
RNA
rpa_rna14 tRNA-Met3_Transfer RNAs
RNA
rpa_rna39 tRNA-Gly2_Transfer RNAs
RNA
rpa_rna42 tRNA-Gly3_Transfer RNAs
RNA
rpa_rna4
tRNA-Thr2_Transfer RNAs
RNA
rpa_rna24 tRNA-Gly1_Transfer RNAs
RNA
rpa_rna15 tRNA-Lys1_Transfer RNAs
RNA
rpa_rna27 tRNA-Leu4_Transfer RNAs
RNA
rpa_rna38 tRNA-Arg5_Transfer RNAs
RNA
rpa_rna46 tRNA-Phe1_Transfer RNAs
RNA
putative indolepyruvate ferredoxin
Energy production and
rpa1224
oxidoreductase, alpha subunit
Conversion
Energy production and
rpa0188
dihydrolipoamide succinyl transferase
Conversion
Energy production and
rpa1991
conserved hypothetical protein
Conversion
Energy production and
rpa4669
putative racemase
Conversion
117
signal %
25.7590
20.2661
12.3260
9.9423
5.5331
3.2917
0.2591
0.1807
0.1667
0.1363
0.1244
0.0873
0.0820
0.0720
0.0714
0.0645
0.0644
0.0635
0.0619
0.0536
0.0476
0.0411
0.0293
0.0292
0.0279
0.0258
0.0258
0.0246
0.0241
0.0197
0.0194
0.0193
0.0189
0.0188
0.0187
0.0156
0.0155
0.0134
0.0115
0.0115
0.0104
0.0102
0.0086
0.0086
0.0298
0.0263
0.0218
0.0211
rpa0672
4-hydroxybenzoyl-CoA reductase, third of
three subunits
rpa3973
cytochrome c556
rpa3215
putative nitroreductase
rpa4822
possible alcohol dehydrogenase
rpa4253
NADH-ubiquinone dehydrogenase chain
M
rpa4566
putative acetate kinase
rpa3392
possible nifU homolog
rpa0219
succinate dehydrogenase membrane
anchor/cytochrome b subunit
rpa0489
ferredoxin II
rpa4181
rpa1911
nicotinamide nucleotide transhydrogenase,
subunit alpha2
Nickel-dependent hydrogenase b-type
cytochrome subunit
rpa3972
putative diheme cytochrome c-553
rpa4410
glycerol-3-phosphate dehydrogenase
rpa2421
NADH:ubiquinone oxidoreductase 17.2 k
rpa1453
putative denitrification protein NorE
rpa1957
alkanal monooxygenase (LuxA-like
protein)
rpa0411
inorganic pyrophosphatase
rpa1437
putative nitrogenase molybdenum-iron
protein alpha chain (nitrogenase
component I) (dinitrogenase)
rpa3709
possible hemoprotein
rpa1136
putative L-lactate permease
prpa2
partition protein
rpa1622
conserved unknown protein
rpa1283
rpa4020
rpa0668
rpa3806
homoserine/homoserine lactone/threonine
efflux protein
possible branched-chain amino acid
transport system permease protein
putative ABC transporter subunit,
substrate-binding component
permease of ABC transporter,putatively for
branched chain amino acuds
rpa3730
GMC-type oxidoreductase
rpa1218
putative ABC transporter periplasmic
protein
118
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Energy production and
Conversion
Cell Division and Chromosome
Partitioning
Cell Division and Chromosome
Partitioning
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
0.0198
0.0188
0.0183
0.0179
0.0174
0.0137
0.0127
0.0124
0.0121
0.0116
0.0116
0.0112
0.0111
0.0110
0.0097
0.0094
0.0093
0.0089
0.0086
0.0085
0.0364
0.0092
0.0493
0.0432
0.0335
0.0302
0.0298
0.0287
rpa0371
rpa4035
rpa1651
rpa1127
rpa3297
rpa0985
rpa4813
putative cystathionine gamma-lyase
possible ABC transport system permease
protein
possible leucine/isoleucine/valine-binding
protein precursor
probable branched-chain amino acid
transport protein AzlC
possible branched-chain amino acid
transport system substrate-binding protein
putative branched-chain amino acid
transport system substrate- binding protein
possible branched chain amino acid
periplasmic binding protein of ABC
transporter
rpa3685
leucine aminopeptidase
rpa4440
putative cyclohexadienyl dehydrogenase
rpa3429
serine acetyltransferase
rpa1798
rpa1445
rpa1741
rpa0587
rpa1655
rpa3725
putative periplasmic binding protein for
ABC transporter for branched chain amino
acids
putative oligopeptide transport
ATP-binding protein
possible branched-chain amino acid
transport system substrate-binding protein
putative cationic amino acid transporter
possible urea/short-chain binding protein
of ABC transporter
possible leucine/isoleucine/valine-binding
protein precursor
rpa2166
conserved hypothetical protein
rpa4179
conserved unknown protein
rpa1479
rpa2040
rpa3436
rpa0666
rpa0106
rpa3722
rpa2277
rpa2408
rpa2463
putative transport system ATP-binding
protein
possible choline ABC transporter
ATP-binding subunit
GCN5-related N-acetyltransferase
putative ABC transporter subunit,
membrane spanning domain +
ATP-binding component
possible branched-chain amino acid
transport system substrate-binding protein
putative branched-chain amino acid
transport system permease protein
possible ABC transporter, permease
protein
putative aliphatic amidase
expression-regulating protein, AmiC
putative cysteine desulfurase, nifS
homolog
119
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
0.0282
0.0262
0.0253
0.0209
0.0205
0.0193
0.0177
0.0177
0.0177
0.0170
0.0159
0.0158
0.0153
0.0153
0.0149
0.0146
0.0145
0.0142
0.0138
0.0130
0.0121
0.0109
0.0108
0.0107
0.0105
0.0104
0.0096
rpa0025
gamma-glutamyltranspeptidase
rpa0658
benzoyl-CoA reductase subunit
rpa0872
putative peptidyl-dipeptidase
rpa1403
possible Glutamine amidotransferase
rpa1890
hypothetical protein
rpa4561
GMP reductase
rpa1005
NUDIX hydrolase
rpa4071
rpa3818
carbamoyl-phosphate synthase large
subunit
phosphoribosylformylglycinamidine
synthase I
rpa1981
ribose 5-phosphate isomerase
rpa3825
conserved hypothetical protein
rpa2169
ribulose-bisphosphate carboxylase-like
protein, rubisco-like protein
rpa1692
putative transmembrane transport protein
rpa4800
possible transporter
rpa2099
Putative diterpenoid Major Facilitator
Superfamily (MFS) transporter
rpa3039
possible polysaccharide deacetylase
rpa3164
possible chitooligosaccharide deacetylase
rpa3781
possible sugar kinase
rpa2341
putative dihydroxy-acid dehydratase
rpa3516
rpa0127
rpa2781
rpa0378
possible bcr efflux pump, Major Facilitator
Superfamily (MFS)
putative permease protein of sugar ABC
transporter
TrapT dctQ-M fusion permease,
dicarboxylate transport
putative alpha-D-galactoside
galactohydrolase
rpa4645
fructose-1,6-bisphosphatase
rpa2395
putative carboxyphosphonoenolpyruvate
phosphonomutase
rpa3311
glycosyl hydrolase
rpa2198
possible multidrug efflux protein
rpa0087
putative Major Facilitator Superfamily
(MFS) transporter
120
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Amino Acid Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Nucleotide Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
0.0095
0.0087
0.0087
0.0231
0.0217
0.0159
0.0128
0.0113
0.0100
0.1641
0.0397
0.0367
0.0175
0.0157
0.0149
0.0137
0.0131
0.0126
0.0126
0.0122
0.0118
0.0110
0.0107
0.0105
0.0101
0.0098
0.0097
0.0094
rpa2271
putative Pyruvate kinase
rpa3634
putative Transaldolase Phosphoglucose
isomerase
rpa1299
conserved hypothetical protein
rpa1257
rpa1028
rpa1507
rpa2089
rpa0300
rpa1506
rpa0930
rpa2671
rpa2094
rpa1723
rpa1520
rpa1546
rpa0875
rpa2918
rpa1603
rpa0531
rpa0659
rpa2138
rpa0818
rpa1710
rpa1030
rpa4699
rpa4651
rpa3075
rpa4002
rpa1411
rpa2185
rpa0335
rpa3441
rpa0484
rpa1699
rpa1614
rpa4553
rpa3349
rpa3305
rpa1973
rpa1835
rpa0816
possible cobalamin (5`-phosphate)
synthase
molybdopterin biosynthesis, protein B
putative magnesium chelatase subunit
BchD
precorrin 2 methylase
putative dephospho-CoA kinase CoaE
putative Mg chelatase subunit Bchl
possible 3-octaprenyl-4-hydroxybenzoate
carboxy-lyase
putative FAD-dependent monoxygenase
putative
nicotinate-nucleotide--dimethylbenzimidaz
ole phosphoribosyltransferase
phenylacetyl-CoA ligase
hydroxyneurosporene methyltransferase
CrtF
Mg-protoporphyrin IX methyl transferase
ferrochelatase
undecaprenyl pyrophosphate synthetase
putative fatty acid desaturase
beta-ketothiolase, acetoacetyl-CoA thiolase
benzoyl-CoA reductase subunit
putative acyl-CoA dehydrogenase
probable 3-hydroxyacyl-CoA
dehydrogenase
putative acyl-CoA dehydrogenase
possible CoA transferase, subunit B
possible dehydrogenase
possible glutaconate CoA-transferase,
subunit A
putative malonyl CoA-acyl carrier protein
transacylase
putative acyl-CoA dehydrogenase
possible enoyl-CoA hydratase/isomerase
nodN-like protein
putative phospholipid N-methyltransferase
enoyl-CoA hydratase/isomerase family
protein
putative enoyl-CoA hydratase
putative acyl-CoA dehydrogenase
isovaleryl-CoA dehydrogenase
putative acyl-CoA dehydrogenase
putative exopolysaccharide biosynthesis
protein
enoyl-CoA hydratase/isomerase family
3-hydroxyisobutyrate dehydrogenase
methylmalonyl-CoA mutase, subunit
alpha, N-terminus
putative acyl-CoA dehydrogenase
121
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
Carbohydrate Transport and
Metabolism
0.0092
0.0091
0.0090
Coenzyme Metabolism
0.0383
Coenzyme Metabolism
0.0310
Coenzyme Metabolism
0.0233
Coenzyme Metabolism
Coenzyme Metabolism
Coenzyme Metabolism
0.0148
0.0133
0.0127
Coenzyme Metabolism
0.0120
Coenzyme Metabolism
0.0116
Coenzyme Metabolism
0.0110
Coenzyme Metabolism
0.0108
Coenzyme Metabolism
0.0107
Coenzyme Metabolism
Coenzyme Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0105
0.0089
0.0382
0.0350
0.0305
0.0221
0.0215
Lipid Metabolism
0.0209
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0205
0.0199
0.0169
Lipid Metabolism
0.0169
Lipid Metabolism
0.0152
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0152
0.0148
0.0143
0.0134
Lipid Metabolism
0.0129
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
Lipid Metabolism
0.0124
0.0118
0.0115
0.0112
Lipid Metabolism
0.0105
Lipid Metabolism
Lipid Metabolism
0.0100
0.0097
Lipid Metabolism
0.0089
Lipid Metabolism
0.0086
rpa0030
Bacterial Sun/eukaryotic nucleolar
Nop1/Nop2
rpa3237
30S ribosomal protein S14
rpa0526
50S ribosomal protein L32
rpa1589
30S ribosomal protein S4
rpa0633
probable ribonuclease p protein component
(protein c5)
rpa3255
30S ribosomal protein S12
rpa0368
possible pseudouridine synthases
rpa0243
putative 16S rRNA processing protein.
rpa2651
Regulator of chromosome condensation,
RCC1:Endoribonuclease L-PSP
rpa1191
putative RNA methyltransferase
rpa3078
30S ribosomal protein S18
rpa3246
30S ribosomal protein S19
rpa1402
putative Glu-tRNA amidotransferase,
subunit A
rpa0490
conserved hypothetical protein
rpa0607
putative protoporphyrinogen oxidase,
hemK protein
rpa0474
conserved unknown protein
rpa3272
50S ribosomal protein L1
rpa0038
ribosomal protein L20
rpa2767
ribosomal protein L13
rpa0051
putative sigma-54 modulation protein
rpa2453
translation peptide releasing factor RF-2
rpa3241
30S ribosomal protein S17
rpa3399
rpa2527
cold shock protein
possible DNA-dependent RNA polymerase
putative nucleoside diphosphate kinase
regulator
transcription termination factor rho
possible transcriptional regulator
putative transcriptional regulator
putative transcriptional regulator
cold shock protein
possible cold shock protein
putative sigma-70 factor, ECF subfamily
possible ArsR protein
rpa0866
rpa0296
rpa0443
rpa0903
rpa3451
rpa3672
rpa1173
rpa1339
rpa3559
122
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Translation, Ribosomal Structure
and Biogenesis
Transcription
Transcription
0.0312
0.0284
0.0244
0.0226
0.0192
0.0186
0.0176
0.0150
0.0150
0.0147
0.0140
0.0135
0.0129
0.0126
0.0121
0.0108
0.0106
0.0103
0.0101
0.0095
0.0092
0.0087
0.0281
0.0256
Transcription
0.0229
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
Transcription
0.0216
0.0211
0.0191
0.0179
0.0178
0.0169
0.0152
0.0147
rpa4448
rpa2222
rpa0828
rpa0982
rpa1958
rpa4098
rpa1954
rpa1019
rpa3718
rpa2294
rpa2994
rpa1008
rpa3791
rpa0680
possible RNA polymerase ECF-type sigma
factor
possible transcriptional regulator
putative transcriptional regulator
putative transcriptional regulator PcaR
possible transcriptional regulator
unknown protein
putative tetR-family transcriptional
regulator
possible transcriptional activator HlyU
putative transcriptional regulator
putative transcriptional regulator
transcriptional regulator, LysR family
possible transcriptional regulator
putative transcriptional regulator
TetR/AcrR family
possible tetR family transcriptional
regulator
rpa4277
conserved hypothetical protein
rpa4546
conserved hypothetical protein
rpa1293
putative IS5 transposase
rpa4082
possible phage-like integrase
rpa4826
DNA helicase
rpa0878
Helix-turn-helix, AraC type
rpa1067
rpa2953
rpa2332
rpa0341
possible 3-methyladenine DNA
glycosylase I
possible DNA-binding protein hu-alpha
(NS2) (HU-2)
Rare lipoprotein A:Staphylococcus
nuclease (SNase-like)
possible Ada polyprotein
(O6-methylguanine-DNA
methyltransferase)
prpa5
resolvase
rpa1332
possible phage integrase/recombinase
rpa2816
excinuclease ABC subunit A
prpa1
replication protein
rpa4541
DNA invertase gene rlgA
rpa0084
formamidopyrimidine-DNA glycosylase
rpa0399
possible transposase
rpa1099
Holliday junction nuclease
rpa4315
conserved hypothetical protein
123
Transcription
0.0144
Transcription
Transcription
Transcription
Transcription
Transcription
0.0140
0.0126
0.0124
0.0117
0.0115
Transcription
0.0114
Transcription
Transcription
Transcription
Transcription
Transcription
0.0114
0.0107
0.0107
0.0106
0.0100
Transcription
0.0097
Transcription
0.0085
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
DNA Replication, Recombination
and Repair
Cell Envelope Biogenesis, Outer
Membrane
0.0777
0.0245
0.0215
0.0199
0.0181
0.0171
0.0166
0.0163
0.0158
0.0153
0.0144
0.0134
0.0131
0.0130
0.0124
0.0109
0.0096
0.0086
0.0646
rpa3536
putative penicillin-binding protein
rpa0222
Beta-Ig-H3/Fasciclin domain
rpa2815
possible outer membrane protein
rpa3482
putative membrane protein
rpa1774
OmpA/MotB domain, possible porin
rpa4502
putative outer membrane protein
rpa3986
putative ADP-heptose--LPS
heptosyltransferase II
rpa2909
lipid-A-disaccharide synthase
rpa3476
possible energy transducer TonB
rpa0879
CBS domain:Sugar isomerase
(SIS):KpsF/GutQ family protein
rpa0076
Nucleotidyl transferase
rpa3145
possible transglycosylase SLT domain
rpa1148
putative sugar transferase
rpa3467
probable UDP-glucose 4-epimerase
rpa2837
Peptidoglycan-binding LysM:Peptidase
M23/M37
rpa0173
putative sugar nucleotide dehydratase
rpa0389
putative penicillin-insensitive murein
endopeptidase A
rpa1960
putative RND efflux membrane protein
rpa0156
tonB like protein
rpa3902
putative curli production
assembly/transport component csgg
precursor
putative periplasmic carboxyl-terminal
processing protease
Flagellar basal body-associated protein
FliL
putative flagellar basal-body rod protein
flgg (distal rod protein)
putative flagellar motor switch protein
possible flagellar motor protein MotB
possible basal-body rod modification
protein flgD
putative flagellar L-ring protein FlgH
rpa3812
putative holocytochrome c synthase
rpa0889
small heat shock protein
rpa3333
rpa0169
rpa3898
rpa3900
rpa1265
rpa0921
rpa0644
124
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
Cell Envelope Biogenesis, Outer
Membrane
0.0469
0.0442
0.0218
0.0172
0.0164
0.0164
0.0155
0.0155
0.0149
0.0136
0.0128
0.0120
0.0110
0.0099
0.0098
0.0097
0.0096
0.0096
0.0095
Cell Envelope Biogenesis, Outer
Membrane
0.0094
Cell Envelope Biogenesis, Outer
Membrane
0.0086
Cell Motility and Secretion
0.0145
Cell Motility and Secretion
0.0134
Cell Motility and Secretion
Cell Motility and Secretion
0.0133
0.0131
Cell Motility and Secretion
0.0109
Cell Motility and Secretion
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
0.0091
0.1003
0.0756
rpa2456
possible bacterioferritin co-migratory
protein
rpa3627
putative glutathione peroxidase
rpa1320
conserved hypothetical protein
rpa2068
conserved unknown protein
rpa1477
putative permease protein
rpa0820
putative glutathionine S-transferase
rpa4084
AAA ATPase
rpa0054
putative small heat shock protein
rpa0876
conserved unknown protein
rpa2604
peptidyl prolyl cis-trans isomerase
rpa3799
DUF182
rpa4011
possible serine protease/outer membrane
autotransporter
rpa2266
putative glutathione S-transferase
rpa1022
possible outer membrane protein
rpa2461
Protein of unknown function UPF0075
rpa2165
chaperonin GroES2, cpn10
rpa4314
possible glutathione S-transferase
rpa2164
chaperonin GroEL2, cpn60
rpa1828
Pyrrolidone-carboxylate/pyroglutamyl
peptidase I (C15)
rpa0934
conserved unknown protein
rpa4069
DUF25
rpa0204
putative heme exporter protein B (heme
ABC transporter membrane protein),
cytochrome c biogenesis
rpa0043
possible glutathione S-transferase
rpa2838
rpa3002
rpa1773
rpa0724
rpa0688
putative D-aspartate protein
carboxylmethyltransferase type II
potassium-transporting atpase c chain,
KdpC
putative DMT superfamily
multidrug-efflux transporter
putative high-affinity nickel-transport
protein
ATP-binding component, PhnN protein,
possible kinase
125
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Posttranslational Modification,
Protein Turnover, Chaperones
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
0.0438
0.0360
0.0353
0.0295
0.0253
0.0202
0.0198
0.0196
0.0173
0.0160
0.0155
0.0148
0.0146
0.0146
0.0142
0.0116
0.0114
0.0113
0.0098
0.0095
0.0093
0.0090
0.0090
0.0085
0.0621
0.0296
0.0267
0.0225
rpa4636
FeoA family
rpa1496
possible monooxygenase
rpa0517
putative transcriptional regulator (Fur
family)
rpa2353
putative nitrogenase NifH subunit
rpa2120
putative hemin binding protein
rpa0691
rpa2793
phosphonate ABC transporter,
ATP-binding component,PhnK protein
pH adaptation potassium efflux system
phaF
rpa4457
putative sulfide dehydrogenase
rpa4732
possible Cation transport regulator protein
rpa2041
rpa2610
ABC-transport protein, ATP-binding
protein
aliphatic sulfonate transport ATP-binding
protein, Subunit of ABC transporter
rpa4635
ferrous iron transport protein B
rpa4501
phnA-like protein
rpa2043
putative ABC transporter, periplasmic
substrate-binding protein
rpa3600
bacterioferritin
rpa2272
conserved unknown protein
rpa2195
possible exopolyphosphatase
rpa1000
rpa0695
rpa0502
rpa0099
rpa4186
rpa2281
rpa2339
rpa1693
rpa3736
rpa3004
rpa1259
Nitrogenase-associated protein:Arsenate
reductase and related
PhnG protein, phosphonate metabolism,
function unknown
probable HlyC/CorC family of transporters
with 2 CBS domains
putative oligopeptide ABC transporter
(ATP-binding protein)
Integral membrane protein TerC family
putative low-affinity phosphate transport
protein
possible iron response transcription
regulator
superoxide dismutase
putative phosphate transport system
substrate-binding protein
potassium-transporting ATPase, A chain,
KdpA
putative cation-transporting P-type ATPase
126
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
Inorganic Ion Transport and
Metabolism
0.0203
0.0199
0.0186
0.0182
0.0174
0.0167
0.0159
0.0159
0.0153
0.0152
0.0145
0.0141
0.0139
0.0134
0.0133
0.0128
0.0127
0.0126
0.0114
0.0114
0.0113
0.0111
0.0111
0.0109
0.0101
0.0100
0.0094
0.0091
rpa2307
possible tonB-dependent receptor
precursor
rpa3465
putative long-chain-fatty-acid CoA ligase
rpa4145
dissimilatory nitrite reductase
rpa1942
putative
2-hydroxyhepta-2,4-diene-1,7-dioate
isomerase
rpa0464
putative long-chain fatty-acid-CoA ligase
rpa3758
2-oxo-hepta-3-ene-1,7-dioate hydratase
rpa3072
3-oxoacyl-acyl carrier protein synthase II
rpa3716
pimeloyl-CoA ligase
rpa1003
putative acyl-CoA synthase
rpa3657
putative long-chain fatty acid-CoA ligase
rpa1242
putative long-chain-fatty-acid CoA ligase
rpa2100
possible hydrolase
rpa3962
ATP-binding component of ABC
transporter
rpa2665
possible acid-CoA ligase
rpa1976
possible TrapT family, dctM subunit,
glutamate transport
rpa4341
Beta-lactamase-like
rpa2417
rpa0632
rpa2681
rpa0078
rpa2811
rpa1940
rpa2373
putative 3-ketoacyl-CoA reductase
60 kDa inner membrane protein
possible lactam utilization protein.
Protein of unknown function UPF0079
possible mazG
putative quinone oxidoreductase
possible outer membrane protein
PAP/25A core domain:DNA polymerase,
beta-like region
rpa0276
127
Inorganic Ion Transport and
Metabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
Secondary metabolites
biosynthesis, transport and
catabolism
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0086
0.0279
0.0211
0.0201
0.0193
0.0158
0.0151
0.0148
0.0135
0.0119
0.0117
0.0116
0.0103
0.0099
0.0091
0.0088
0.0880
0.0571
0.0447
0.0375
0.0346
0.0317
0.0279
0.0268
rpa0224
rpa2151
rpa1155
rpa3223
rpa0709
rpa4811
rpa0044
rpa1596
rpa0810
rpa2201
rpa0909
rpa1914
rpa2105
rpa3459
rpa4577
rpa2004
rpa2809
rpa0198
rpa2597
rpa1095
rpa0449
rpa4300
rpa2259
rpa3918
rpa4424
rpa3417
rpa2224
rpa3508
rpa1080
rpa3801
rpa1856
rpa3866
rpa3461
rpa0209
rpa1698
rpa0532
rpa4509
rpa0324
rpa4528
rpa2303
rpa2812
similar to eukaryotic molybdopterin
oxidoreductase:Tat pathway signal
Permeases of the drug/metabolite
transporter (DMT) superfamily
putative pmbA protein, maturation of
antibiotic MccB17
putative alginate lyase
conserved unknown protein
conserved hypothetical protein
bacitracin resistance protein
possible tryptophan synthase beta chain
Permeases of the drug/metabolite
transporter (DMT) superfamily
quinone oxidoreductase
Trp repressor binding protein
possible glycohydrolase
non-heme chloroperoxidase
possible TrapT family, fused dctM-Q
subunits, C4-dicarboxylate transport
putative permease protein of sugar ABC
transporter
conserved hypothetical protein
Beta-lactamase
conserved hypothetical protein
probable nfrA protein
Beta-lactamase-like
possible hydrolases/phosphatases
Permeases of the drug/metabolite
transporter (DMT) superfamily
possible arsH protein
conserved hypothetical protein
Beta-lactamase-like
Lipocalin-related protein and Bos/Can/Equ
allergen:Rhomboid-like protein
possible trbI, a component on a type IV
secretion system (Y10832)
ATP-binding component of ABC
transporter
possible 2-pyrone-4,6-dicarboxylate
hydrolase
MoxR-like ATPases
Protein of unknown function UPF0050
conserved unknown protein
conserved hypothetical protein
putative cell division protein FtsY
DUF81
beta-ketothiolase, acetoacetyl-CoA
reductase
possible TrapT family, fused dctQ-M
subunits, C4-dicarboxylate transport
Uroporphyrin-III C/tetrapyrrole
(Corrin/Porphyrin) methyltransferase
conserved hypothetical protein
putative 3-hydroxyacyl-CoA
dehydrogenase
probable HlyC/CorC family of transporters
with 2 CBS domains
128
General Function Prediction Only
0.0258
General Function Prediction Only
0.0248
General Function Prediction Only
0.0232
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0231
0.0221
0.0216
0.0215
0.0212
General Function Prediction Only
0.0208
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0199
0.0196
0.0193
0.0192
General Function Prediction Only
0.0187
General Function Prediction Only
0.0179
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0167
0.0165
0.0162
0.0159
0.0157
0.0150
General Function Prediction Only
0.0148
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0146
0.0141
0.0141
General Function Prediction Only
0.0141
General Function Prediction Only
0.0138
General Function Prediction Only
0.0136
General Function Prediction Only
0.0135
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0133
0.0132
0.0129
0.0128
0.0128
0.0127
General Function Prediction Only
0.0126
General Function Prediction Only
0.0122
General Function Prediction Only
0.0117
General Function Prediction Only
0.0114
General Function Prediction Only
0.0114
General Function Prediction Only
0.0112
rpa0471
rpa0556
rpa1705
rpa1878
rpa4365
rpa2101
rpa1737
rpa0214
rpa0451
rpa3439
rpa1540
rpa0586
rpa0642
rpa3377
rpa3605
rpa0302
rpa0830
rpa1697
rpa0728
rpa3619
rpa1328
rpa3412
rpa0162
rpa2288
rpa3770
rpa1106
rpa2170
rpa4217
rpa2290
rpa1133
rpa3131
rpa4192
rpa2470
rpa1659
rpa4137
rpa2732
rpa1007
rpa1186
rpa4141
rpa3108
rpa1119
rpa1623
rpa3804
rpa1747
rpa0530
rpa1203
rpa2401
rpa2548
rpa2024
rpa2820
rpa1330
putative creatine amidohydrolase
HNH endonuclease:HNH nuclease
possible 3-hydroxyacyl-CoA
dehydrogenase type II
putative 6-aminohexanoate-dimer
hydrolase
GCN5-related N-acetyltransferase
conserved hypothetical protein
possible dehydrogenase
hypothetical protein
putative RimI protein, peptide
N-acetyltransferase
putative hydrolase
Coenzyme B12-binding
short shain alcohol dehydrogenase
conserved hypothetical protein
3-oxoacyl-acyl carrier protein reductase
trans-aconitate methyltransferase
putative protein-export protein SecB
conserved unknown protein
Competence-damaged protein
conserved unknown protein
putative vanillate O-demethylase
oxygenase, iron-sulfur subunit
possible histidine kinase
Patatin-like phospholipase domain
possible GTP-binding proteins
Streptomyces cyclase/dehydrase
conserved unknown protein
conserved hypothetical protein
hypothetical protein
conserved unknown protein
conserved hypothetical protein
unknown protein
conserved hypothetical protein
conserved hypothetical protein
Protein of unknown function,
HesB/YadR/YfhF
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
possible 2,3-dihydroxyphenylpropionate
1,2-dioxygenase
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
putative TolA
conserved unknown protein
conserved unknown protein
conserved hypothetical protein
conserved unknown protein
conserved hypothetical protein
conserved unknown protein
conserved hypothetical protein
conserved unknown protein
DUF433
conserved hypothetical protein
129
General Function Prediction Only
General Function Prediction Only
0.0108
0.0108
General Function Prediction Only
0.0107
General Function Prediction Only
0.0105
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0105
0.0102
0.0101
0.0098
General Function Prediction Only
0.0098
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
0.0096
0.0096
0.0096
0.0094
0.0092
0.0091
0.0091
0.0090
0.0089
0.0089
General Function Prediction Only
0.0088
General Function Prediction Only
General Function Prediction Only
General Function Prediction Only
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0087
0.0087
0.0085
0.1118
0.0461
0.0456
0.0411
0.0383
0.0371
0.0315
0.0291
0.0270
Function Unknown
0.0265
Function Unknown
Function Unknown
Function Unknown
0.0251
0.0247
0.0203
Function Unknown
0.0189
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0186
0.0185
0.0182
0.0176
0.0171
0.0165
0.0157
0.0155
0.0142
0.0140
0.0137
0.0135
0.0135
0.0130
rpa2058
rpa3726
rpa1885
rpa4418
rpa2025
rpa1907
rpa3109
rpa1604
rpa0827
rpa4628
rpa4774
rpa0152
rpa4499
rpa2295
rpa0036
rpa4613
rpa1311
rpa4142
rpa0031
rpa3041
rpa3511
rpa4357
rpa0571
rpa2381
rpa2409
rpa0377
rpa4312
rpa1533
rpa4173
rpa1965
rpa4816
rpa0145
rpa4686
rpa3546
rpa4233
rpa4009
rpa0852
rpa3101
rpa2076
rpa2903
rpa3380
rpa4781
rpa0052
rpa3434
conserved hypothetical protein
conserved unknown protein
putative portal protein, R. capsulatus GTA
orfg3 homologue
conserved unknown protein
possible MgtC Mg2+ transport protein
putative protein
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
Protein of unknown function,
HesB/YadR/YfhF
conserved unknown protein
Protein of unknown function, UPF0066
unknown protein
unknown protein
conserved unknown protein
DUF683
possible 4-carboxymuconolactone
decarboxylase
PilT protein, N-terminal
conserved hypothetical protein
conserved hypothetical protein
ErfK/YbiS/YcfS/YnhG
conserved unknown protein
two-component, response regulator
probable transmembrane sensor involved
in iron transport regulation
possible AmiR antitermination protein
conserved unknown protein
putative signal-transduction sensor protein
tryptophan-rich sensory protein
GGDEF
possible Adenylate cyclase
putative two-component sensor protein
putative two-component system, response
regulator.
possible ABC transporter, periplasmic
amino acid-binding protein
possible methyl-accepting chemotaxis
protein (MCP) s HAMP domain
possible K+ channel
GAF domain
probable two-component system, response
regulator
conserved unknown protein
possible sensory transduction histidine
kinase
SOS response regulator lexA protein
putative chemotaxis protein cheY3
possible phosphate regulon,
two-component sensor histidine kinase,
phoR
putative nitrogen regulatory IIA
protein(enzyme IIA-NTR)
Universal stress protein (Usp)
130
Function Unknown
Function Unknown
0.0124
0.0123
Function Unknown
0.0123
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0122
0.0121
0.0117
0.0116
0.0115
0.0111
Function Unknown
0.0109
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
0.0102
0.0097
0.0095
0.0095
0.0095
0.0093
Function Unknown
0.0093
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Function Unknown
Signal Transduction Mechanisms
0.0092
0.0091
0.0089
0.0087
0.0085
0.0436
Signal Transduction Mechanisms
0.0244
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
Signal Transduction Mechanisms
0.0162
0.0158
0.0157
0.0149
0.0136
0.0133
0.0127
Signal Transduction Mechanisms
0.0126
Signal Transduction Mechanisms
0.0126
Signal Transduction Mechanisms
0.0119
Signal Transduction Mechanisms
Signal Transduction Mechanisms
0.0116
0.0113
Signal Transduction Mechanisms
0.0112
Signal Transduction Mechanisms
0.0110
Signal Transduction Mechanisms
0.0104
Signal Transduction Mechanisms
Signal Transduction Mechanisms
0.0098
0.0094
Signal Transduction Mechanisms
0.0094
Signal Transduction Mechanisms
0.0093
Signal Transduction Mechanisms
0.0090
rpa2299
rpa4329
rpa2050
rpa1309
rpa0768
rpa0864
rpa1066
rpa4805
rpa4574
rpa3780
rpa3975
rpa0505
rpa0684
rpa1491
rpa0116
rpa2091
rpa0618
rpa4239
rpa4224
rpa1289
rpa3334
rpa3787
rpa2603
rpa3400
rpa3361
rpa0771
rpa3180
rpa4003
rpa1499
rpa1526
rpa4128
rpa3877
rpa1832
rpa0259
rpa4344
rpa3367
rpa0117
rpa1114
rpa2246
rpa2059
rpa4525
rpa3030
rpa4292
rpa1492
rpa1846
rpa1717
possible bacterial regulatory proteins, luxR
family
conserved unknown protein
conserved hypothetical protein
possible transposase
possible transposase
hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical protein
hypothetical protein
light harvesting protein B-800-850, beta
chain E (antenna pigment protein, beta
chain E) (LH II-E beta)
possible dTDP-D-glucose-4,6-dehydratase
hypothetical protein
unknown protein
conserved unknown protein
unknown protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
possible protein commonly found in
insertion elements
hypothetical protein
conserved hypothetical protein
hypothetical protein
light-harvesting complex 1 alpha chain
conserved hypothetical protein
hypothetical protein
unknown protein
Histidine kinase, HAMP region
hypothetical protein
possible activator of photopigment and puc
expression, appA-like
dTDP-D-glucose 4,6-dehydratase
conserved unknown protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
light harvesting protein B-800-850, alpha
chain B (antenna pigment protein, alpha
chain B) (LH II-B alpha)
light harvesting protein B-800-850, alpha
chain E (antenna pigment protein, alpha
chain E) (LH II-E alpha)
unknown protein
hypothetical protein
131
Signal Transduction Mechanisms
0.0090
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.2180
0.1839
0.1042
0.0781
0.0517
0.0450
0.0428
0.0374
0.0369
0.0361
0.0347
0.0346
not in COGs
0.0339
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0315
0.0293
0.0292
0.0289
0.0286
0.0283
0.0271
0.0257
0.0256
0.0253
0.0248
not in COGs
0.0247
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0244
0.0241
0.0238
0.0238
0.0236
0.0231
0.0227
0.0224
0.0220
not in COGs
0.0218
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0217
0.0211
0.0211
0.0205
0.0205
0.0202
not in COGs
0.0202
not in COGs
0.0201
not in COGs
not in COGs
0.0200
0.0200
rpa2654
rpa4100
rpa1157
rpa1455
rpa4228
rpa2254
rpa2023
rpa3196
rpa4291
rpa4804
rpa3214
rpa3859
rpa4393
rpa1111
rpa3648
rpa4535
rpa2885
rpa1422
rpa2188
rpa2991
rpa1847
rpa1528
rpa2860
rpa4230
rpa4127
rpa0528
rpa4532
prpa3
rpa3035
rpa3874
rpa3438
rpa0486
rpa0543
rpa0538
rpa2721
rpa0008
rpa3013
rpa0608
rpa0960
rpa1525
rpa0092
rpa3005
rpa0778
rpa1842
rpa3158
rpa4080
rpa3197
rpa2747
rpa3601
rpa1621
light harvesting protein B-800-850, beta
chain A (antenna pigment protein, beta
chain A) (LH II-A beta)
hypothetical protein
conserved unknown protein
nitric-oxide reductase subunit C
hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
light harvesting protein B-800-850, beta
chain B (antenna pigment protein, beta
chain B)
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
hypothetical protein
conserved hypothetical protein
photosynthetic reaction center M protein
hypothetical protein
conserved unknown protein
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
BsuBI-PstI family restriction endonuclease
hypothetical protein
hypothetical protein
hypothetical protein
Staphylococcus nuclease (SNase-like)
unknown protein
putative Omp2b porin
hypothetical protein
circadian clock protein
light harvesting protein B-800-850, beta
chain D (antenna pigment protein, beta
chain D) (LH II-D beta)
conserved unknown protein
uptake hydrogenase regulatory protein
hupV pseudogene, frameshifted
light-harvesting complex 1 beta chain
conserved hypothetical protein
hypothetical protein
conserved hypothetical protein
conserved unknown protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
hypothetical protein
132
not in COGs
0.0195
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0194
0.0194
0.0194
0.0192
0.0191
0.0189
0.0185
not in COGs
0.0184
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0178
0.0178
0.0178
0.0177
0.0175
0.0172
0.0164
0.0163
0.0163
0.0162
0.0161
0.0159
0.0158
0.0157
0.0157
0.0157
0.0154
0.0153
0.0152
0.0152
0.0151
0.0150
0.0150
0.0148
0.0145
0.0144
0.0144
not in COGs
0.0143
not in COGs
0.0142
not in COGs
0.0141
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0140
0.0140
0.0139
0.0138
0.0137
0.0136
0.0135
0.0133
0.0133
0.0133
0.0131
rpa1714
rpa4349
rpa2803
rpa2419
rpa3875
rpa4831
rpa0967
rpa4713
rpa3373
rpa1475
rpa0890
rpa0522
rpa3541
rpa4319
rpa0834
rpa1646
rpa2250
rpa3928
rpa1197
rpa1516
rpa4473
rpa0114
rpa4007
rpa2547
rpa2900
rpa4384
rpa0871
rpa3103
rpa2334
rpa1952
rpa1342
rpa1286
rpa4714
rpa4735
rpa4183
rpa2494
rpa0922
rpa4063
rpa3854
rpa3152
rpa1964
rpa4210
rpa2813
rpa2444
rpa3142
rpa1041
rpa2653
rpa1279
rpa1893
rpa2509
rpa1308
rpa3328
hypothetical protein
conserved unknown protein
hypothetical protein
possible outer membrane protein, possible
porin
conserved unknown protein
conserved hypothetical protein
hydrogenase expression/formation protein
hupF
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
possible activator of photopigment and puc
expression
hypothetical protein
hypothetical protein
putative CoxF
hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
possible rifampin ADP-ribosyl transferase
hypothetical protein
conserved hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical membrane protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
unknown protein
conserved hypothetical protein
conserved hypothetical protein
light harvesting protein B-800-850, alpha
chain A (antenna pigment protein, alpha
chain A) (LH II-A alpha)
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
conserved hypothetical protein
133
not in COGs
not in COGs
not in COGs
0.0129
0.0129
0.0128
not in COGs
0.0127
not in COGs
not in COGs
0.0127
0.0127
not in COGs
0.0125
not in COGs
not in COGs
not in COGs
not in COGs
0.0124
0.0123
0.0122
0.0120
not in COGs
0.0118
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0117
0.0116
0.0116
0.0115
0.0115
0.0115
0.0114
0.0114
0.0113
0.0113
0.0110
0.0110
0.0110
0.0110
0.0109
0.0109
0.0109
0.0108
0.0107
0.0107
0.0106
0.0106
0.0106
0.0103
0.0103
0.0102
0.0102
0.0101
0.0100
0.0100
0.0100
0.0099
0.0099
0.0099
not in COGs
0.0098
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0098
0.0097
0.0095
0.0094
0.0094
rpa1345
rpa2228
rpa1962
rpa4720
rpa2028
rpa0801
rpa4273
rpa1859
rpa2658
rpa0566
rpa0366
rpa4375
rpa1495
rpa2338
rpa3998
conserved hypothetical protein
probable trbK
unknown protein
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
hypothetical protein
conserved hypothetical protein
hypothetical protein
conserved unknown protein
unknown protein
unknown protein
unknown protein
unknown protein
hypothetical protein
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
not in COGs
0.0094
0.0094
0.0093
0.0093
0.0092
0.0091
0.0091
0.0091
0.0090
0.0090
0.0089
0.0088
0.0088
0.0087
0.0086
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%.
134
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GENERAL DISCUSSION
In natural environments, cells were often experimented sudden nutrient
depletion. However how they adapt those starvation conditions and how relate the
energy status of starved cells to survivability were not clear. In this study, to clarify the
relationship between energy status and survivability under the non-growing conditions,
survivability of purple non-sulfur photosynthetic bacteria were investigated.
Purple non-sulfur photosynthetic bacteria are distributed throughout the alpha
and beta subgroups of Proteobacteria and they are widely distributed in natural
environments. All species used in this study, which are belonging to representative
different orders, maintained high survivability under carbon starvation conditions in the
light. These suggest that purple bacteria can survive by using light energy even when
carbon source is depleted. This survival strategy is probably common in purple
photosynthetic bacteria and may also contribute to their widely distribution in natural
environments.
Metabolomic and transcriptomic analysis were performed to clarify the
physiological characteristics of the starved cells in Rhodopseudomonas palustris
CGA009. In the illuminated starved cells, the high amount of ATP was detected, and
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. The change of
long-chain fatty acid composition became remarkable compared to that in the dark.
These results suggested that ATP produced by illumination was utilized to
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macromolecule turnover and starvation response such as change of lipid composition
was different depending on the energy states of the starved cells. High expression of
many genes related to protein turnover in the light also supports this idea. The starved
cells both in the light and dark included considerable amount of mRNA; it was
suggested that active gene expression still occurred in the starved cells even when
energy supply was very low in the dark. In addition, global gene expressions of the
starved cells in the light and dark were clearly different; the starved cells in the light
were characterized to express genes related to protein turnover, while the starved cells
in the dark were characterized to express genes related to inorganic ion transporters. It
was suggested that these different patterns of gene expression were reflected the
starvation strategy corresponding to energy status of starved cells.
Rhodopseudomonas palustris CGA009, which showed longer survivability
under starvation conditions in the dark than other purple bacteria, showed higher
resistance against osmotic stresses. High survivability under hypertonic stress
conditions and gene expression under the dark suggested that the high survivability of R.
palustirs CGA009 may be related to maintaining cytoplasmic homeostasis. Their
survivability may be affected their habitat and their survival strategy; 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 including various soil.
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