Rhodobacter capsulatus is a nonsulfur purple photosynthetic bacterium that exhibits diverse respiratory abilities, allowing this organism to grow under a variety of environmental conditions. Branched respiratory electron transport pathways allow
R. capsulatusto grow aerobically in the dark, either chemoautotrophically or chemoheterotrophically, by using O
2 as the terminal electron acceptor. Indeed, its high capacity for aerobic chemoautotrophic growth distinguishes it from other well-studied nonsulfur purple bacteria, such as
R. sphaeroidesand
Rhodospirillum rubrum (
26). Like other organisms of this group,
R. capsulatus can also grow anaerobically in the light, either photoautotrophically or photoheterotrophically, using cyclic photosynthetic electron transport to generate a proton motive force. These organisms can grow fermentatively as well. Due to such metabolic versatility,
R. capsulatus provides an excellent system with which to gain insight into the control of redox homeostasis. Yet, compared to the thorough and well-characterized redox control studies of
Escherichia coli (for a review, see reference
17 or
51 and references therein), knowledge of the control of redox homeostasis in
R. capsulatus is somewhat limited.
During phototrophic growth, various electron acceptors are employed, in a hierarchical manner, to maintain a balanced redox state in
R. capsulatus (
50). In the presence of organic carbon under light anaerobic growth conditions (photoheterotrophic growth), the redox-balancing mechanism(s) consists primarily of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway (CBB system). Under some growth conditions, the dinitrogenase enzyme complex (dinitrogenase system), the dimethyl sulfoxide (DMSO) reductase (DMSOR) system, or other systems yet to be identified or implicated in redox control are employed. Specific reactions of the CBB pathway allow CO
2 to function as a sink for excess reducing equivalents generated by the metabolism of carbon substrates such as
l-malate and succinate. Thus, the predominant role of the CBB pathway during photoheterotrophic growth is to balance the oxidation-reduction potential of the cell (
13,
24,
55). The capacity for CO
2-dependent growth under photoautotrophic growth conditions is accomplished primarily by the CBB system, where the chief role of the CBB pathway is to provide the cell with carbon via the assimilation of CO
2. The duality of roles of the CBB system leads to an interplay between the maintenance of redox poise and the control of carbon metabolism under photoheterotrophic and photoautotrophic growth conditions. The dinitrogenase system is synthesized in most phototrophs when the organism is placed in an ammonia-free environment. This system enables
Rhodobacter to grow under conditions in which dinitrogen is the sole source of nitrogen (N
2-dependent growth); i.e., the cells catalyze the reduction and assimilation of atmospheric dinitrogen to ammonia, accompanied by the reduction of protons to molecular hydrogen. The process of dinitrogen fixation requires much reducing power and is an energy-intensive process (
5). Not only does the dinitrogenase system play a primary role in nitrogen metabolism (
23), but it has also been shown to be involved in redox homeostasis in
Rhodobacter and
Rhodospirillum rubrum (
22,
50). Photoheterotrophic growth with a poor nitrogen source such as glutamate signals the cell to synthesize the dinitrogenase system (for a review, see reference
25 and references therein). Under such growth conditions, the excess reducing equivalents generated by the oxidation of carbon substrates, such as malate, are consumed by the reduction of protons and consequent evolution of molecular hydrogen by a hydrogenase-like activity of the dinitrogenase system. This allows the cell to balance its intracellular redox potential (
20). Physiological studies have shown that a link between carbon metabolism and nitrogen metabolism exists that is intimately associated with the control of intracellular redox poise in
R. capsulatus (
50),
R. sphaeroides (
22,
43), and
R. rubrum (
22). Specifically, in the absence of a functional CBB system (achieved through the inactivation of genes encoding key and unique enzymes of the CBB pathway), spontaneous variants of strains with photoheterotrophic competency (PHC) and CBB deficiency dissipate excess reducing equivalents as H
2 gas by derepressing the dinitrogenase system (
22,
50). Respiration of the auxiliary oxidant DMSO or trimethylamine-
N-oxide (TMAO) through the DMSOR system has also been shown to play an important role in the maintenance of redox poise during phototrophic growth of
R. capsulatus(
27,
44). Indeed, DMSO respiration allows growth of CBB-deficient strains of
R. sphaeroides(
11,
18,
19,
55) and
R. capsulatus(
40,
50) under photoheterotrophic growth conditions in the presence of a fixed nitrogen source. Thus, the reduction of DMSO or TMAO serves as an additional mechanism by which to dissipate excess reducing equivalents generated by carbon metabolism.
In this study, reporter-gene promoter fusions were employed to examine the expression of the CBB, dinitrogenase, and DMSOR systems in response to different environmental and metabolic signals. CBB-deficient strains and a dinitrogenase-derepressing strain of R. capsulatus were used in these studies. Contributions of the different redox systems to photoautotrophic carbon metabolism and the interaction with nitrogen metabolism were explored, and the results of these studies reflect on the overall control of redox homeostasis inR. capsulatus.
DISCUSSION
Nonsulfur purple bacteria couple their ability to assimilate carbon dioxide and dinitrogen to photosynthetic energy generation and the production of required reducing equivalents (
15). However, knowledge of how various redox-balancing systems interact and contribute to successful photoheterotrophic or photoautotrophic metabolism is limited. In the present study, the expression of three important redox-balancing mechanisms, the CBB, dinitrogenase, and DMSOR systems, was shown to be either coordinately regulated or influenced by the presence of one system or the other. This is necessary to ensure balance in the use of reducing equivalents generated by phototrophic metabolism (Fig.
5). The control of anaerobic respiratory pathway gene expression in
R. capsulatus is comparable to the situation in
E. coli, where there is also coordinate and integrative control over the redox-balancing systems (for a review, see reference
17 and references therein). However, in the present study, evidence for linkage in the control of key redox-balancing systems (i.e., those important for CO
2 fixation, nitrogen fixation, and DMSO respiration) is presented for the first time in both the photoheterotrophic and photoautotrophic growth modes.
The interplay between the dual roles (maintenance of redox poise and carbon metabolism) of the CBB system has been shown to correlate with the expression of the DMSOR system. The integration of the CBB system and the DMSOR system in phototrophic metabolism is not unprecedented. Phototrophic growth on highly reduced substrates such as butyrate and propionate is known to depend upon the addition of exogenous CO
2 as an electron acceptor (
52). Under these conditions, the CBB system is obligately required for growth (
11,
12,
39). An auxiliary oxidant, DMSO or TMAO, can substitute for CO
2 under these phototrophic growth conditions (
44). Moreover, during phototrophic growth on less-reduced carbon substrates (e.g.,
l-malate), the DMSOR system can also replace the need for a functional CBB system in
R. capsulatus (
40,
50). The results of the present investigation indicated that
cbbI of the CBB system of
R. capsulatus was responsive to activation of the DMSOR system under photoautotrophic growth conditions, while
cbbII was unaffected by the DMSOR system under either photoheterotrophic or photoautotrophic growth conditions. By contrast, RubisCO-deficient strain SBI/II exhibited a different response in that both
cbbI and
cbbII promoter activities were raised to photoautotrophic (1.5% CO
2–98.5% H
2) wild-type levels under photoheterotrophic growth conditions in the presence of DMSO. In fact, all of the redox systems, as exemplified by the respective promoter fusions, were up-regulated in strain SBI/II.
In
Rhodobacter, redox homeostasis is achieved through the interplay of cyclic photosynthetic electron transport and specific redox-balancing mechanisms of anaerobic metabolism during phototrophic growth (
30). It has been suggested that the electron acceptors involved in photosynthetic metabolism function as a sink for excess reducing equivalents or prevent the overreduction of the cyclic electron transport system. This interaction between redox poise and electron transport occurs at the level of the ubiquinone pool (
13; Fig.
5). Respiratory electron flow to the DMSOR system has been shown to branch from cyclic electron transport at the level of the ubiquinone pool (
27,
28); thus, activation of the DMSOR system under phototrophic growth conditions may siphon reductant from the ubiquinone pool. Studies of the related organism
R. sphaeroides indicated that flux from the ubiquinone pool is transduced through a pathway involving
cbb3-type cytochrome
c oxidase, while a signal involved in the flow of reductant is conveyed to the PrrBA (RegBA) signal transduction pathway (
33-35). In
R. capsulatus, the two-component signal transduction system RegBA (PrrBA) has been shown to be involved in the regulation of operons important for photosynthetic gene expression (
46), CO
2 fixation (
53), and nitrogen fixation and H
2oxidation (
8,
50). Indeed, the current model suggests that the RegBA system responds to the overall intracellular redox state (
22,
49), although more-detailed studies are required to elucidate the specific redox-sensing mechanisms that influence the Reg system of
R. capsulatus (
4,
8). Under photoautotrophic growth conditions in
R. capsulatus, the RegBA global regulatory system was shown to be involved in activation of
cbbI promoter expression, as well as maximal expression of the
cbbII promoter (
53). It is possible that by activating the DMSOR system, which alters the oxidation-reduction potential of the ubiquinone pool, a redox signal is transmitted to a regulatory system that, in turn, controls the expression of key operons involved in phototrophic metabolism. In
R. capsulatus, the RegBA system plays more of a critical role in regulating
cbbI since this operon is up-regulated only during photoautotrophic metabolism while
cbbII is expressed under a variety of conditions. This could explain the sensitivity of
cbbI to activation of the DMSOR system under photoautotrophic growth conditions. Alternatively, additional, unknown factors that have been postulated to be involved in expression of the CBB system in
R. sphaeroides (
6,
7) and
R. capsulatus (
53) could play a critical role in transmitting a redox signal to control key operons involved in redox homeostasis.
During photoheterotrophic metabolism, redox poise is also achieved by the coordinate integration of the DMSOR and CBB systems, as well as the dinitrogenase system. Indeed, in the absence of an operational CBB system, spontaneous variants of
R. capsulatusderepress the dinitrogenase system, resulting in photoheterotrophic competency (
50). Dinitrogenase-catalyzed proton reduction and the consequent evolution of H
2 gas are important for maintenance of redox poise in
R. capsulatus (
20). The current study monitored the interplay between the CBB and dinitrogenase systems, as well as the DMSOR system, in CBB-deficient and PHC strains of
R. capsulatus. Although the specific regulatory mechanism(s) involved in the derepression of dinitrogenase in PHC mutant strains of
R. capsulatus remains to be established, it should be noted that the PrrBA (RegBA) two-component regulatory system is involved in the maintenance of the PHC phenotype of an
R. sphaeroides dinitrogenase-derepressing strain (
22). Additionally, the Reg system was shown to be involved in the control of nitrogen fixation in wild-type
R. capsulatus (
8) and
Bradyrhizobium japonicum (
3).
The specific integration of redox mechanisms with the derepression of the dinitrogenase system in
R. capsulatus differs from the situation in
R. sphaeroides(
50). For example, activation of the DMSOR system under photoheterotrophic growth conditions diminishes
nifexpression in a dinitrogenase-derepressing strain of
R. capsulatus while exhibiting no effect in
R. sphaeroides (
22,
42). We have also observed differences in
R. sphaeroides and
R. capsulatus cbbI promoter expression in an
R. capsulatus PHC strain background. This could be due to differences in general redox response between
cbbIs of the two organisms, the differential effects of specific metabolic signals on the cognate CbbR proteins, or a combination of both possibilities. With different ecological niches in aquatic ecosystems (
41), it is not unexpected that
R. capsulatus and
R. sphaeroides differentially regulate processes involved in the control of redox homeostasis in response to the environmental milieu. An indication of this possibility was previously suggested by the demonstration of differences in the roles of the global regulatory systems of FnrL (
47,
59,
60) and RegBA (PrrBA) (
2,
9,
10,
32,
46) during phototrophic growth in
R. sphaeroides and
R. capsulatus.
A question that must be addressed concerns the potential role and coordinate control of specific metabolic signals with redox homeostasis in response to environmental factors. In
R. sphaeroides (
7) and
Rhodopseudomonas palustris, whose genomic sequence was recently completed (
http://www.jgi.doe.gov/tempweb/JGImicrobial/html/index.html ), it is apparent that a single
cbbR gene controls the transcription of the two major
cbb operons. A separate upstream and divergently transcribed
cbbR gene, however, controls each
cbb operon in
R. capsulatus (
38,
39,
53). LysR-type transcriptional regulators, such as CbbR, generally utilize a metabolite or coinducer produced by the pathway they regulate (
45). Clearly, the current study has demonstrated that a complex interrelationship of specific redox-balancing systems exists in
R. capsulatus and probably other nonsulfur purple bacteria. Since activation of the DMSOR system affects control of the CBB system under photoautotrophic environmental conditions and in some instances may also cause up-regulation of promoter sequences important for redox balancing under photoheterotrophic growth conditions, it is important to determine if these observed regulatory events are coordinated with the appearance of and subsequent interaction with a specific metabolic signal metabolite(s). Perhaps strain SBI/II can be effectively used in such investigations since there is dramatic up-regulation of operons important for redox control in this strain. Continued studies of the nature of the signal(s) that influences both CbbR and the more global redox-sensing pathways required for photoheterotrophic and photoautotrophic growth are warranted.