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Microbiology and Molecular Biology Reviews, June 2004, p. 263-279, Vol. 68, No. 2
1092-2172/04/$08.00+0     DOI: 10.1128/MMBR.68.2.263-279.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

RegB/RegA, a Highly Conserved Redox-Responding Global Two-Component Regulatory System

Sylvie Elsen,^1, Lee R. Swem,^2, Danielle L. Swem,^2, and Carl E. Bauer^2*

Laboratoire de Biochimie et de Biophysique des Systèmes Intégrés (UMR 5092 CNRS-CEA-UJF), CEA-Grenoble, 38054 Grenoble Cedex 9, France,¹ Department of Biology, Indiana University, Bloomington, Indiana 47405²

SUMMARY

INTRODUCTION

RegB/RegA TWO-COMPONENT REGULATORY SYSTEM

Discovery

Homologous Systems

SENSOR KINASE RegB

Kinase Activity

Phosphatase Activity

Redox-Sensing Capabilities

In vivo redox control.

In vitro rcdox control.

Other input signals.

RESPONSE REGULATOR RegA

Effect of Phosphorylation

DNA-Binding Sites

Transcription Regulation

Reg REGULON

Photosynthesis

Electron Transfer System

Aerobic Respiration

Anaerobic Respiration

Carbon Fixation

Nitrogen Fixation

Denitrification

Hydrogen Oxidation

Dehydrogenases

Aerotaxis

CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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RegB and RegA were originally described as cognate members of a two-component signal transduction system that are involved in anaerobic synthesis of the photosystem in Rhodobacter capsulatus (55, 78). However, it is now known that RegB and RegA constitute a highly conserved global regulatory system that provides an overlying layer of redox control on a variety of energy-generating and energy-utilizing biological processes. Specifically, photosynthesis, carbon fixation, nitrogen fixation, hydrogen oxidation, denitrification, aerobic and anaerobic respiration, electron transport, and aerotaxis are now known to be part of the RegB/RegA regulon.

In addition to studies revealing the extent of the RegB/RegA regulon, there has been a substantial amount of biochemical research on mechanistic functions of these regulators. RegB, a histidine sensor kinase, has been characterized in terms of kinetics, necessary components, and conditions needed to observe redox-regulated in vitro autophosphorylation. RegA has also been studied in terms of the effect of phosphorylation on DNA-binding activity, transcriptional modulation and interactions with other regulators. In addition, a consensus RegA DNA-binding motif has been identified.

The R. capsulatus RegB/RegA system has provided the groundwork for the discovery and characterization of homologues found in a wide variety of photosynthetic and nonphotosynthetic bacteria. Among -proteobacteria, there is an unprecedented 100% conservation of the helix-turn-helix (HTH) DNA-binding motif of RegA homologues, suggesting that there are significant constraints on the ability of RegA to undergo genetic drift (see Fig. 1). Additionally, a variety of RegB homologues show conservation of a "redox box" that is at least partially responsible for controlling RegB kinase activity in response to changes in environmental redox conditions (see Fig. 2). Below we describe our current understanding of this multivalent system.

View larger version (90K): [in this window] [in a new window]   FIG. 1. Alignment of RegA homologues present in genome databases. The input domain contains a conserved phosphate-accepting aspartate residue, denoted by a star. The output domain contains the three-helical bundle DNA-binding domain, denoted by 6, 7, and 8. The flexible linker region, which connects the input and output domains, is labeled Hinge. The alanine responsible for the RegA* phenotype is indicated by an octagon. Red residues represent 100% conserved amino acids, blue residues represent 50% or higher conserved amino acids, and yellow residues represent conservative amino acid changes.

View larger version (217K): [in this window] [in a new window]   FIG. 2. Alignment of RegB homologues present in genome databases. Membrane-spanning domains are indicated as regions TM1 through TM6. The site of histidine phosphorylation is denoted by a star within the H-box. The threonine residue important for phosphatase activity is indicated by a square. The location of the redox-active cysteine is denoted by a circle within the redox box. The ATP-binding domains are indicated as the N, G1, F, and G2 boxes. The color scheme is as described in the legend to Fig. 1.

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Sequence analysis indicated that regB and regA genes from most species are localized to a similar region of the chromosome. In R. capsulatus, R. sphaeroides, R. sulfidophilum, and R. denitrificans, the regB gene is divergently transcribed from a putative senC-regA-hvrA operon (10, 29, 30, 31, 53, 55). A variation occurs in Caulobacter crescentus, where a putative regB-regA operon is transcribed divergently from senC. The other organisms listed in Fig. 1 and 2 have regB and regA genes linked in a single putative regB-regA operon without senC or hvrA genes being found within close proximity on the chromosome (details of SenC and HvrA are discussed below).

As shown in the sequence alignments of RegB (Fig. 2) and RegA (Fig. 1) homologues, RegB and RegA proteins are highly conserved among photosynthetic and nonphotosynthetic - and -proteobacteria. The RegA homologues can be placed into two groups based on the sequence similarity of their output domain. Group 1 members have 100% conservation of the HTH DNA-binding motif, while group 2 homologues have a similar but nonidentical HTH region (Fig. 1). Overall, group 1 homologues exhibit a remarkable 78.7 to 84.2% sequence identity to RegA from R. capsulatus, with 93% overall identity in the C-terminal output domain. This level of conservation suggests that there are significant constraints on the variability of RegA to undergo mutational change in these species. In contrast to extensive analysis of group 1 homologues, there has been limited characterization of group 2 homologues. Mutational analysis of roxR, which is a group 2 RegA homologue in P. aeruginosa, indicates that RoxR controls respiratory gene expression in P. aeruginosa as does the RegB/RegA system in R. capsulatus (15, 83). Interestingly, several photosynthetic and nonphotosynthetic species, such as C. cresentus, R. capsulatus, and Mesorhizobium loti, contain both group 1 and group 2 homologues (Fig. 1).

Among RegB homologues, there are also two identifiable groups composed of similar members, as discussed for RegA homologues (Fig. 2). Differences between group 1 and group 2 RegB homologues occur in the regions between transmembrane 6 and the H box (amino acids 245 to 269), between the redox box and the N box (amino acids 335 to 355), and at the carboxyl terminus (amino acid 455 to end) (Fig. 2).

In addition to RegB and RegA proteins having a high level of sequence identity, they have functional similarity. For example, Emmerich et al. (25) demonstrated that RegB and RegA homologues from R. capsulatus, B. japonicum, and S. meliloti are interchangeable. More precisely, phosphotransfer was observed between the B. japonicum RegB homologue (RegS) and R. capsulatus RegA. The RegA homologue from B. japonicum (RegR) was shown to bind to the R. capsulatus puf and puc operon promoters. R. capsulatus RegA, and its homologue from S. meliloti (ActR), can activate transcription of the fixR nifA operon from B. japonicum in vivo (25). Furthermore, Comolli and Donohue (15) demonstrated that RegA homologues from R. sphaeroides (PrrA) and P. aeruginosa (RoxR) also succeeded in heterologous complementation. These studies demonstrate that homologous Reg proteins are interchangeable in vitro as well as in vivo.

Mutational analysis of several -proteobacterial RegA homologues indicates that they control a similar set of target genes such as respiratory and nitrogenase genes, details of which are discussed below. Thus, it appears that the RegB/RegA system is involved in global regulation in many diverse species of bacteria.

SENSOR KINASE RegB

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The R. capsulatus regB gene encodes a 460-amino-acid (50.1-kDa) protein that is composed of two domains; a N-terminal transmembrane domain and a C-terminal cytoplasmic "transmitter" domain that typifies histidine protein kinases (Fig. 2). The presence of six hydrophobic membrane-spanning regions in the N terminus was predicted by hydrophobicity profile topological modeling and confirmed by alkaline phosphatase fusions to both R. capsulatus and R. sphaeroides RegB homologues (11, 68). Regions of conservation in the transmitter domain include the H-box of the dimerization domain, containing the conserved site of autophosphorylation (His225), and the N, G1, F, and G2 boxes, which define the nucleotide-binding cleft (11, 30, 55, 68, 82).

Whereas the transmitter domain constitutes the catalytic phosphorylation domain, the role of the amino-terminal transmembrane domain is still under investigation. Insertion of RegB into the membrane has been suggested to enhance the dimerization of the protein and/or be important for regulation of the catalytic activities of RegB (41). Comparisons of in vitro properties of N-terminally truncated cytosolic and full-length proteins indicate that the transmembrane domain may play a regulatory role (74). Furthermore, a mutant with a mutation in the R. sphaeroides RegB homologue in the cytoplasmic loop between the second and third membrane-spanning domains (leucine 78 to proline) exhibits constitutive (oxygen-insensitive) kinase activity in vivo (30). There is also a significant region of conservation located in the second periplasmic loop between the third and fourth transmembrane domain of RegB that could constitute a potential redox-sensing component of this protein.

The first demonstration of phosphotransfer from R. capsulatus RegBP to RegA was reported by Inoue et al. (41). Phosphotransfer studies showed that the transfer of phosphate from the cytosolic domain of RegB to RegA in vitro is rapid (<1 min) (7). Similar phosphotransfer kinetics have been reported by investigators using truncated cytosolic and full-length versions of R. sphaeroides RegB homologues. No back-transfer of phosphate from RegAP to RegB has been observed (14, 74). A soluble truncated form of the RegB homologue (RegS_C) in B. japonicum was shown to autophosphorylate at residue His219 in the presence of [-³²P]ATP and to transfer the phosphate to the Asp63 residue of the RegA homologue (RegR) (26). Since phosphotransfer is very rapid, autophosphorylation of RegB appears to be the rate-limiting step in the phosphorylation of RegA.

Because both truncated and full-length RegB exhibit the same phosphatase activity, it is assumed that modulation of phosphatase activity does not require the N-terminal domain of RegB (74). However, further studies are required to determine if phosphatase activity is redox regulated.

In the E. coli sensor kinase EnvZ, it is apparent that a threonine residue positioned 4 residues downstream of the conserved phosphorylated histidine is important for phosphatase activity (42). This threonine residue is positioned on the same -helical face directly below the phosphorylated histidine. It is thought that the histidine residue may deprotonate the threonine hydroxyl group to form a good nucleophile that attacks the phosphoryl group bound to the aspartate residue of the cognate response regulator. Interestingly, RegB homologues also contain a 100% conserved threonine residue located 4 residues downstream of the phosphorylated histidine (Fig. 2), suggesting that RegB may exhibit a similar phosphatase mechanism with RegA. Nonetheless, Potter et al. (74) point out that a significant difference between RegB and EnvZ phosphatase activity exists, given that EnvZ has a requirement for ATP or a nonhydrolyzable analogue as a cofactor for phosphatase activity while RegB lacks this nucleotide requirement.

In vivo redox control. One redox signal that has been proposed to regulate RegB is the redox state of the aerobic respiratory chain (29, 31, 43, 60, 64, 76). This conclusion is based on the observation that mutations that disrupt R. sphaeroides and R. capsulatus cytochrome cbb₃ oxidase lead to elevated aerobic expression of RegB/RegA-regulated genes (9, 29). It has therefore been suggested that cytochrome cbb₃ oxidase generates an "inhibitory" signal that represses the RegB/RegA two-component system. This signal may either inhibit the kinase activity or stimulate the phosphatase activity of RegB, which, in turn, controls the amount of phosphorylated RegA (60, 63). It has also been proposed that electron flow through the cytochrome cbb₃ oxidase could be monitored by CcoQ, the smallest subunit of cbb₃ oxidase that is thought to stabilize the CcoP subunit from proteolytic degradation under aerobic conditions (66). In this model, CcoQ would function as a "transponder," relaying an inhibitory signal to RegB (63). This model, however, must be viewed with caution since it has also been reported that there is no effect of disrupting the ccoNOQP genes (cytochrome cbb₃ oxidase) on RegA-dependent expression of the two cbb promoters in R. capsulatus (33). The same investigators also observed no effect on cytochrome cbb expression after disruption of the ccoQ gene in R. sphaeroides (33). Clearly, additional experimentation is needed to sort out the role of cytochrome cbb₃ oxidase, if any, on controlling the activity of RegB.

Genetic studies also implicate SenC (also called PrrC) in the transduction of a "redox signal" in R. sphaeroides and in R. capsulatus. Specifically, inactivation of senC (prrC), which is cotranscribed with regA, results in an oxygen-insensitive phenotype in R. sphaeroides (29, 31, 43, 60, 64, 76). The senC gene encodes a 23.2-kDa membrane-spanning protein containing a highly conserved motif, CPDVCP, with the cysteine residues thought to be involved in copper binding (54). Interestingly, SenC also has sequence similarity to a family of oxidoreductases that are involved in disulfide bond oxidation and reduction (54). One possibility is that SenC could be directly involved in modulation of the oxidation and reduction of a redox-active cysteine residue within RegB (84). Furthermore, eukaryotic homologues of SenC (called ScoI) are thought to be associated with cytochrome c oxidase assembly (35), so it has also been proposed that a redox signal could be passed from cytochrome cbb₃ oxidase to RegB via SenC (64).

It is also interesting that RegA controls expression of the regB gene, the senC-regA-hvrA operon, and the ccoNOQP operon that codes for cytochrome cbb₃ oxidase (19, 83). Hence, not only does the RegB/RegA system down-regulate its own synthesis, but it also controls transcription of components that may modulate its own activity.

In vitro rcdox control. Initially, the in vitro kinase activity of cytosolic truncated RegB was shown to be unaffected by redox changes (7, 14, 41). It was therefore assumed that the redox-sensing ability of RegB lay within its transmembrane-spanning region. Although there may indeed be a role for the membrane-spanning region in redox sensing in vivo, recent analysis indicates that the cytosolic domain of RegB also plays a critical role in controlling the autophosphorylation activity of the kinase. An advance in our molecular understanding of the control of RegB activity occurred when Swem et al. (83, 84) demonstrated that RegB requires the coordination of a metal, such as copper, to respond to redox. This crucial component was not observed previously because of the addition of the metal chelator, EDTA, to the protein purification mixtures. Additionally, it was demonstrated that RegB contains a redox-responsive Cys (Cys265) located in the dimerization interface, more precisely in the "redox box" (Fig. 2), which seems to be involved in the redox-sensing mechanism of RegB (84). It is proposed that this Cys residue undergoes modification such as oxygen-dependent formation of a sulfenic acid (Cys-S-OH) and/or disulfide bond formation in vivo that inhibits kinase activity. In vitro disulfide bond formation was shown to require the presence of a divalent metal ion, with ligand coordination of the metal playing a structural role necessary to align RegB monomers into a conformation allowing the redox-active Cys residue to function properly (84).

A copper requirement for RegB in vivo is also supported by the observation that mutations in the hypothetical copper ATPase-type transporter protein, RdxI, and a copper oxidase, RdxB (CcoG), which is involved in intracellular oxidation of Cu⁺ to Cu²⁺ (47), lead to constitutive RegB autophosphorylation as assayed by measuring the elevated aerobic expression of genes under control of the RegB/RegA system (61, 76). The involvement of an intermolecular disulfide bridge in the control of RegB activity is also supported by an increase in in vitro RegB phosphorylation in the presence of dithiothreitol (74).

In vitro analysis indicates that an intermolecular disulfide bond forms between RegB dimers, resulting in a tetramer when oxidized and shutting off kinase activity (84). However, the redox state of this cysteine in vivo has not been elucidated. Nonetheless, substitution of alanine for this cysteine leads to significantly elevated aerobic expression of RegB/RegA-targeted genes in vivo, indicating its importance for redox sensing.

Other input signals. Although the redox-responding properties of the RegB/RegA system are well established, a recent study by Abada et al. (1) has revealed that the R. capsulatus system may also respond to other stimuli, specifically a flow of intermediates through the bacteriochlorophyll (Bchl) biosynthetic pathway. Their study demonstrated that synthesis of the pigment-binding proteins encoded by the RegB/RegA-regulated puf and puc operons is reduced when Bchl biosynthesis is defective. They further demonstrated that Bchl-dependent expression of puf and puc depends on the RegB/RegA system, since disruption of either gene affects Bchl-dependent transcription. Interestingly, a regA mutant does not sense the absence of Bchl, whereas a regB mutant does. One possibility is that RegA directly or indirectly senses the amounts of photopigments being synthesized through its receiver domain.

RESPONSE REGULATOR RegA

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R. capsulatus RegA is a 184-amino-acid 20.4-kDa protein containing several highly conserved residues that are typically found in two-component response regulators. Conserved residues include a phosphate-accepting aspartate and an "acid pocket" containing two highly conserved aspartate residues in the N-terminal receiver domain (Fig. 1). The receiver domain is linked by a four-proline hinge to a 50-amino-acid C-terminal output domain that contains a three-helix bundle HTH DNA-binding motif (18, 49, 78). As discussed above, RegA homologues are highly conserved among numerous -proteobacterial species, with an unprecedented complete conservation of the DNA-binding domain.

The inherent stability of the aspartyl-phosphate among the RegA homologues, as a measure of half-life, is high relative to that observed with other response regulators. The half-life was about 90 min for R. capsulatus RegAP (7), about 162 min for the RegA homologue (RegRP) from B. japonicum (26), and 330 min for R. sphaeroides RegAP (14). RegA from R. sphaeroides has also been shown to use the small phosphodonor acetylP in vitro (14). Autophosphorylation of R. capsulatus RegA in the presence of acetylP was also reported by Hemschemeir et al. (39).

DNase I footprint analysis of RegA binding to the puc promoter region revealed that phosphorylated and unphosphorylated wild-type RegA, and RegA* protect identical regions with different affinities. This observation indicates that phosphorylation does not affect the points of protein-DNA interaction but, rather, affects the affinity for the binding site (7). Indeed, phosphorylation is reported to increase the DNA-binding affinity of RegA by at least 16-fold. Interestingly, unphosphorylated RegA* exhibits DNA-binding activity comparable to that of phosphorylated wild type, supporting in vivo evidence that RegA* is able to promote transcription activation even in the absence of phosphotransfer from RegB. A further six-fold increase in the DNA-binding activity of RegA* is achieved upon phosphorylation (7). These results are consistent with what was reported for the B. japonicum RegA homologue (RegR), whose DNA-binding activity is increased by at least eightfold on phosphorylation (26). Phosphorylation-induced stimulation of DNA-binding activity was also reported for P. aeruginosa RoxR (15). However, Hemschemeier et al. (40) have reported the same affinity (K_D, 50 to 70 nM) for phosphorylated and unphosphorylated RegA when using the puc promoter as a probe in vitro. The reason for this discrepancy is not clear. In addition, they showed that deletion of the last 13 amino acids, which contain the DNA-binding motif of RegA, inhibited DNA-binding ability in vitro as well as the activation of puc and puf expression in vivo. This confirms the importance of DNA binding for the transcriptional activation activity of RegA.

Analysis of mutations at the site of phosphorylation (Asp63) in R. capsulatus RegA, as well as with RegA homologues from B. japonicum and R. sphaeroides, has been performed. A D63N mutation in RegR of B. japonicum rendered the protein unable to be phosphorylated and also unable to bind DNA, as demonstrated by gel retardation experiments with a fixR-nifA promoter probe (26). In contrast, experiments involving a D63K RegA mutant from R. capsulatus showed that the mutant protein was capable of binding DNA, despite an inability to be phosphorylated. This indicated that phosphorylation might not affect the DNA-binding ability but, rather, might facilitate a conformational change allowing appropriate interactions of RegA with RNA polymerase (38). This theory was further supported by in vitro transcription assays by Comolli et al. (14), involving wild-type PrrA and a D63A mutant of PrrA from R. sphaeroides. Their studies revealed that both unphosphorylated and phosphorylated wild-type PrrA are able to activate the transcription of cycA P2, with phosphorylated PrrA exhibiting greater activity than unphosphorylated PrrA. Interestingly, the D63A form of PrrA was unable to activate any detectable amounts of transcription. These data suggest that phosphorylation of Asp63 and the mere presence of Asp63 may affect several steps of activation such as the DNA binding of RegA and the interaction of RegAP with RNA polymerase.

Superimposed on the effect of phosphorylation are numerous in vivo observations that unphosphorylated RegA is also capable of affecting transcription. Specifically, mutational analysis indicates that phosphorylated RegA functions as an anaerobic repressor of cytochrome cbb₃ oxidase expression while unphosphorylated RegA functions as an aerobic activator of cytochrome cbb₃ oxidase (83, 85). In addition, both phosphorylated and unphosphorylated RegA are involved in activation of ubiquinol oxidase expression (85), activation of cbb operon expression (75), and repression of hupSLC expression (23). Additional DNA-binding studies with both phosphorylated and unphosphorylated RegA are clearly needed to obtain an understanding of the mechanism of activation or repression by unphosphorylated RegA.

The number of RegA DNA-binding sites on target promoters ranges between 1 and 6, based on DNase I protection assays (Fig. 3). Individual sites in the promoter regions have different affinities, as determined by the amount of RegA needed to obtain half-maximal protection of individual sites (83). This finding leads to a classification of RegA-regulated promoters into three types based on the location of the RegA-binding sites. The first type, exemplified by the R. capsulatus puf promoter, has RegA DNA-binding sites that extend from bp –22 to –80 relative to the start site of transcription (Fig. 3). The second type, comprising the R. capsulatus puc, nifA2, cydAB, ccoNOQP, and cbb_II promoters and the R. sphaeroides cbb_I, cbb_II, and cycA promoters, possesses several DNA-binding sites that extend from approximately bp –50 to –890 upstream from the transcriptional start site. The third type, represented by the R. capsulatus cbb_I, petABC, cycA, and cycY promoters, contains one site overlapping the start site of transcription (from bp –19 to +22) as well as DNA-binding sites from bp –50 to –225. Although it is possible that additional DNA-binding sites have been left unrevealed, these different binding locations may allow RegA to interact with RNA polymerase in more than one manner at these promoters. This is especially true in comparisons of promoters that have RegA DNA-binding sites at the region from bp +20 to –50 with promoters that have binding sites just in the region from bp –300 to –890. For example, RegA binding to the puc promoter is centered at around –60, which presumably favors the interaction of RegA with the subunit of RNA polymerase (18). In contrast, RegA binds to the region of the puf promoter from bp –22 to –52 (18), which presumably favors interaction with the subunit of the RNA polymerase. There is also RegA binding to the region of the puf promoter from bp –68 to –80, which would favor an additional interaction with the subunit of the RNA polymerase. These interactions should overcome the presence of poor ⁷⁰ recognition sequences harbored by the puf promoter, which uses the ⁷⁰ housekeeping RNA polymerase (8). Clearly, additional detailed analyses of the mechanism of RegA activation and repression at these different classes of promoters is warranted, and indeed needed, to better understand how RegA interacts with RNA polymerase and controls gene expression.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Location of RegA-binding sites (black boxes) that have been located by DNase I footprint protection analysis. The location of transcription initiation is indicated by an arrow. R. sph., R. sphaeroides; R. caps., R. capsulatus.

A nuclear magnetic resonance (NMR) spectral structure was recently solved for the DNA-binding output domain of RegA bound to DNA (49). This structure demonstrated that the RegA DNA-binding domain is composed of a three-helix bundle encompassing an HTH motif (49). The three helices within the three-helix bundle are labeled in Fig. 1 as 6, 7, and 8, with 7 and 8 comprising the predicted DNA-binding HTH motif. This structure is similar to the three-helix bundle found in the E. coli Fis family of DNA-binding proteins (49). This DNA-binding effector domain of RegA is more structurally similar to Fis than to the effector domain of the response regulator, NtrC (49). The NMR structure has also provided new insights into the DNA recognition and DNA-binding ability of RegA. As demonstrated previously by DNase I protection assays and SELEX (systemic evolution of ligands by exponential enrichment) RegA has a distinct affinity for a GCG...CGC palindromic sequence (28, 83). The NMR structure of a RegA-DNA cocomplex has confirmed that the RegA consensus recognition sequence consists of YGCGRCR_xTATA_xGNCGC (with x denoting a variable number of bases) (49). Surprisingly, the GCG inverted repeat can be separated by a 3- to 9-nucleotide AT-rich spacer region (49). This AT-rich spacer region could presumably allow for DNA bending, which aids RegA recognition and binding (49). The NMR structure clearly identifies the 8 helix and the DNA recognition helix with this helix binding specifically to the GCG inverted repeat sequence within the major DNA groove. The 6 helix appears to undergo nonspecific interactions with the DNA phosphate backbone (49). These data provide a very revealing picture of how RegA recognizes and binds to the numerous promoters under RegB/RegA transcriptional control.

As detailed in the following section, it is a common occurrence that other transcription factors regulate operons found within the RegB/RegA regulon. For instance, RegA regulates the transcription of photosystem promoters in concert with the histone-like proteins, integration host factor (IHF), and HvrA (10, 46, 58), as well as with CrtJ (8, 24, 72, 73) and AerR (17) (details of these additional transcription factors are covered in references 3 through 5). The occurrence of additional regulators of genes that are also controlled by RegA is common. Thus, RegA constitutes a global regulator that provides an overarching layer of redox control on operons that have additional transcription factors that respond to photosynthesis, respiratory electron transport, organic carbon, fixed nitrogen, hydrogen, etc.

Reg REGULON

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As is the case for many RegB/RegA-regulated promoters, numerous other transcription factors have been found to control puf, puh, and puc operon expression in R. capsulatus (3, 4). DNase I footprint assays showed that CrtJ and RegA compete for binding to overlapping sites in the puc promoter (8). An additional aerobic repressor called AerR also represses the puf operon; the site of AerR repression has not yet been determined (17). In addition to CrtJ and AerR, the H-NS-like protein HvrA regulates the puf and puh promoters, where it functions as an activator in R. capsulatus and as a repressor in R. sphaeroides (10, 59, 80). The location of HvrA binding in R. sphaeroides is unknown, but in R. capsulatus, the HvrA DNA-binding site is adjacent to RegA DNA-binding sites (48). In both R. capsulatus and R. sphaeroides, the histone-like protein IHF is required for maximal puc operon transcription (58, 96). FnrL in R. sphaeroides also anaerobically activates puc expression, whereas inactivation of fnrL in R. capsulatus has no noticeable affect on photosynthetic growth (reviewed in reference 96). Thus, there are numerous potential protein-protein interactions that might exist between RegA and a number of other transcription factors that control these photosystem promoters. Currently, little is known about the nature of these interactions or their importance in controlling photosystem gene expression.

In addition to controlling the synthesis of light-harvesting and reaction center apoproteins, RegA affects the expression of the bchE promoter in R. sphaeroides, which encodes an enzyme in the bacteriochlorophyll biosynthesis pathway (62). There are also reports that RegA from R. sphaeroides controls the expression of hemA, hemZ, and hemN (62, 65), which encode one -aminolevulinic acid (ALA) synthase isoenzyme and two coproporphyrinogen III oxidases, respectively: enzymes involved in the Mg- and Fe-tetrapyrrole biosynthetic pathways. Abada et al. (1) have also reported an involvement of the R. capsulatus RegB/RegA system in bacteriochlorophyll-dependent expression of the puc, puf, and bchC operons. Therefore, not only does RegB/RegA control the synthesis of the photosystem and cytochrome apoproteins (the role of RegB/RegA in cytochrome biogenesis is discussed below), but also they control synthesis of bacteriochlorophyll and heme that are bound by these respective apoproteins.

Swem et al. (83) demonstrated that RegB/RegA controls synthesis of cytochrome c₂ as well as cytochrome c_y and the cytochrome bc₁ complex in R. capsulatus. The expression patterns of these different cytochrome genes were compared in wild-type and regA-disrupted strains, which revealed that RegA activates the biosynthesis of cytochromes bc₁ and c₂ under anaerobic, semiaerobic, and aerobic growth conditions, whereas it activates cytochrome c_y only under semiaerobic and anaerobic conditions. DNase I protection assays also demonstrated that RegA binds to two sites on the promoter of the pet (bc₁) operon and to four sites on the promoters of the cycA and cycY genes encoding cytochrome c₂ and cytochrome c_y, respectively (83) (Fig. 3). These three promoters belong to the type III class of RegA-regulated promoters (discussed above), where there is one DNA-binding site that overlaps the start site of transcription and one site located just upstream from the –35 promoter region. The in vivo involvement of these different RegA-binding sites still needs to be determined.

Differences in expression patterns of these various cytochromes suggest that additional transcription factors may also regulate the expression of these genes. For example, evidence suggests the involvement of another protein besides RegA, that regulates cycA expression in the absence of O₂ in R. sphaeroides (45). There is also a response regulator and a putative repressor located just upstream of the pet operon, which is suspected to be involved in controlling the expression of cytochrome bc₁ apoproteins (91). Thus, as with other cellular processes, RegB/RegA appears to be just one component of a more complex regulatory network that controls the biosynthesis of cytochrome apoproteins.

Both of the terminal oxidases are maximally synthesized under semiaerobic growth conditions. However, the two oxidases are differentially regulated in regards to high and no (or very low) oxygen levels, with the ubiquinol oxidase exhibiting a low level of expression under aerobic conditions and a higher level under anaerobic conditions. The converse is true for cytochrome cbb₃ oxidase, which exhibits higher expression under aerobic than anaerobic growth conditions (83, 85). This expression pattern indicates that cytochrome cbb₃ oxidase from R. capsulatus may have a lower affinity for oxygen than does the ubiquinol oxidase (87, 95).

RegA has been observed to activate cytochrome cbb₃ oxidase expression semiaerobically and aerobically while repressing expression anaerobically. The mechanism and the significance of this observation are not yet well understood (83). DNase I footprint analysis revealed that RegA directly controls the synthesis of cytochrome cbb₃ oxidase by binding to a site on the ccoNOQP promoter located just upstream from the –35 sequence (Fig. 3) (83). Although there was an early report that FnrL does not affect cytochrome cbb₃ oxidase synthesis in R. capsulatus (96), more recent analysis indicates that expression of ccoNOPQ is indeed lower in an fnrL-disrupted mutant under semiaerobic and anaerobic conditions (85).

The expression pattern of the cydAB operon, encoding ubiquinol oxidase, suggests that the enzyme has a higher affinity for oxygen than does cytochrome cbb₃ oxidase (83, 85). As observed for the ccoNOQP operon, RegB/RegA is involved in the regulation of cydAB expression, with RegA being required for activation of cydAB transcription under all growth conditions tested. DNase footprint assays indicate that RegA binds to two sites upstream from the –35 region of the promoter (Fig. 3) (83).

Recently, RegB/RegA homologues from P. aeruginosa (RoxS/RoxR) were reported to be involved in the control of aerobic respiration (15). More precisely, RoxS/RoxR controls the induction of the cyanide-insensitive oxidase in the presence of cyanide. It is proposed that RoxR coregulates the cioAB promoter with another anaerobic regulator, ANR, thereby permitting the integration of different stimuli in the control of cyanide-insensitive oxidase expression.

Enzymes of the Calvin cycle are encoded by the cbb_I and cbb_II operons. Transcription of these operons is regulated in response to carbon by the transcriptional activator CbbR, which is a member of the LysR family of transcription factors. CbbR is absolutely required for expression of the cbb_I operon, since inactivation of the R. sphaeroides cbbR gene leads to the absence of transcription through the cbb₁ operon and to a strong reduction of cbb_II expression (34). CbbR directly regulates cbb_I expression by binding to two sites located in the promoter-proximal region, as demonstrated by in vitro DNase I footprint experiments (20, 21).

An involvement of the RegB/RegA system in the biosynthesis of Calvin cycle enzymes was first discovered in R. sphaeroides by screening for mutants that derepress an alternative CO₂ fixation pathway (75). From studies of these mutants, it was demonstrated that RegB (PrrB) of R. sphaeroides was required for positive regulation of the cbb operons, both anaerobically in the light and aerobically in the dark (75). Using purified R. capsulatus RegA*, Dubbs et al. (20, 22) demonstrated that RegA directly controls R. sphaeroides cbb expression by binding to four sites in the cbb_I promoter and to six sites on the cbb_II promoter (Fig. 3). The authors hypothesized that the locations of RegA binding could allow direct interactions with CbbR and/or with RNA polymerase. Furthermore, binding of RegA to the two sites located in the upstream activating sequence in the cbb_I promoter appears responsible for a RegA-mediated 41-fold enhancement in cbb_I expression (20).

Gibson et al. (33) demonstrated that chemoautotrophically grown regA (prrA) mutants of R. sphaeroides differentially express the two cbb operons with expression of the cbb_II promoter being severely reduced and expression of the cbb_I promoter being enhanced in the prrA mutant strain. This result indicates that PrrA functions as an activator of cbb_II and a repressor of cbb_I. Analysis of promoter mutants suggests that RegA may bind to distinct regions in cbb_II and in cbb_I during photoautotrophic and chemoautotrophic growth.

In R. capsulatus, the RegB/RegA system also controls the expression of the two cbb operons that are present in this species (93). The cbb_I and cbb_II operons are regulated by cognate CbbR proteins encoded by the cbbR_I and cbbR_II genes. The cbbR_I and cbbR_II genes are located upstream of, and are divergently transcribed from, the cbb_I and cbb_II operons, respectively. The CbbR_I protein is able to control its own expression (down regulation) as well as cbb_II expression under certain conditions (69, 93). On both the cbb_I and cbb_II operon promoters, CbbR binds to a site that overlaps the –35 region, which suggests that protein-protein interactions between CbbR and the RNA polymerase are required for transcriptional activation. Inactivation of regA and regB affects cbb_I and cbb_II expression, with only 14 and 10% of wild-type levels, respectively, found in a regA-disrupted strain under photoautotrophic growth conditions. RegA* was also shown to bind to two DNA-binding sites in both the cbb_I and cbb_II promoter regions. There is a major high-affinity RegA binding site located at bp –102 to –121, upstream of the cbb_I transcription start site, that is assumed to be involved in transcriptional activation in concert with CbbR_I. A low-affinity RegA binding site is located at positions –4 to –19, overlapping a CbbR_I DNA-binding site located at positions –18 to –79. Presumably the low-affinity RegA binding site that overlaps the CbbR site plays a negative role as a result of RegA-mediated occlusion of CbbR_I binding to this region. On the cbb_II promoter, there are two high-affinity RegA-binding sites, one located at positions –101 to –116 and the second located at positions –124 to –169. The upstream location of these binding sites suggests that they are involved in activation. The cbb_II promoter also contains a CbbR_II DNA-binding site located at positions –19 to –78.

It has been reported that RegA homologues from B. japonicum (RegR) and S. meliloti (ActR) also function as activators of cbb operons in concert with CbbR (27, 32). Thus, as was demonstrated for R. capsulatus, RegA homologues appear to control CO₂ fixation in a number of photosynthetic and nonphotosynthetic bacteria.

Elsen et al. (23) shed light on the mechanism of derepression of nitrogenase in R. capsulatus by showing that the RegB/RegA system indirectly controls expression of the nifHDK operon, which encodes the molybdenum-containing nitrogenase complex. In R. capsulatus and in many other species, nitrogenase expression is regulated by nitrogen limitation through the NtrB/NtrC two-component system. Under nitrogen-limiting conditions NtrB phosphorylates NtrC, which then activates nifA transcription by binding to two tandem sites centered >100 bp upstream of the transcriptional start site. NifA then activates the expression of numerous nif genes, including nifHKD (reviewed in reference 52). In R. capsulatus, there are two functional copies of nifA, nifA1 and nifA2, either of which can activate nifHDK expression. Elsen et al. (23) demonstrated that RegA binds to the nifA2 promoter between the tandem NtrC DNA-binding sites and the –35 and –10 promoter sequences. Interestingly, RegA-mediated activation of nifA2 transcription requires NtrC, indicating that RegAP alone is not sufficient to stimulate nifA2 expression (23). Thus, RegA appears to provide an overarching layer of redox control on top of the control of nitrogen availability provided by NtrC.

In B. japonicum, the RegB/RegA homologues (RegS/RegR) are required for the aerobic and anaerobic expression of the fixRnifA operon (6). Interestingly, a mutation that disrupts the response regulator RegR reduces fixR nifA expression and consequently nitrogen fixation activity. However, no related phenotype was observed on disruption of the sensor kinase, RegS. RegR mutants of B. japonicum form nodules, but the nodules are functionally incapable of fixing nitrogen (a Fix^– phenotype). Electron micrographic analysis indicates that nodules formed on infection by the RegR-disrupted strain failed to produce bacteroids, which are differentiated B. japonicum cells that undertake nitrogen fixation (6).

H₂ regulation is mediated by the two-component regulatory system HupT/HupR, with the response regulator HupR directly activating hupSLC transcription in the presence of H₂ (16). The nonphosphorylated form, HupR, binds to the hupSLC promoter at a palindromic sequence centered at bp –157 with respect to the transcription start site. Maximal expression of hupSLC also requires the binding of IHF between the HupR and RNA polymerase DNA-binding sites (reference 92 and references therein).

Elsen et al. (23) demonstrated that RegA is involved in repressing hupSLC expression under both aerobic and anaerobic heterotrophic growth conditions. A major DNA-binding site of RegA was shown to be located close to the –35 promoter recognition sequence, with a second, lower-affinity RegA-binding site overlapping the IHF DNA-binding region. At that location, it is possible that RegA could prevent either the RNA polymerase or the IHF protein, or both, from binding to the hupSLC promoter. Interestingly, a deletion in RegB can be suppressed by addition of multiple copies of the sensor kinase HupT (36). Presumably, increased amounts of HupT are capable of phosphorylating RegA in the absence of RegB.

In S. meliloti, the RegB and RegA homologues, ActS and ActR, control the biosynthesis of three dehydrogenases, formaldehyde dehydrogenase, formate dehydrogenase, and methanol dehydrogenase, as well as CO₂ fixation (32). The ActS/ActR system is also involved in acid tolerance (90).

It will take further analysis to determine the exact role of RegB and RegA in controlling aerotaxis, such as the ability of RegB to directly affect phosphorylation of the chemotaxis cascade or simply modulate the abundance of the chemosensory apparatus. Nevertheless, aerotactic control going through the RegB/RegA cascade is certainly novel.

CONCLUDING REMARKS

Top References

 
Genetic and biochemical analyses have revealed that the RegB/RegA system is a major global regulator of numerous energy-generating and energy-utilizing cellular processes. The systems controlled by RegB/RegA in R. capsulatus and R. sphaeroides include such fundamental and diverse processes as photosynthesis, CO₂ fixation, N₂ assimilation, hydrogen utilization, denitrification, dehydrogenases, electron transport, and aerotaxis (Fig. 4). The Reg regulon is continuously growing, and it is likely that there are many more, yet to be discovered, target genes under the control of RegB/RegA in these metabolically diverse bacteria.

View larger version (35K): [in this window] [in a new window]   FIG. 4. Diagram of the various RegB/RegA-controlled systems that have been identified in R. capsulatus and R. sphaeroides. UQH2, reduced ubiquinol; UQ, oxidized ubiquinone; FGSH, S-formylglutathione; HMGSH, hydroxymethylglutathione; DMS, dimethyl sulfate.

The observation that highly conserved RegB and RegA homologues exist in many other bacterial species also indicates that the RegB/RegA system constitutes an important redox control element that is not easily replaced by other regulators. Although many questions regarding the function of RegB and RegA in other systems still need to be addressed, evidence is mounting that they control a similar set of target genes in a number of bacterial species.

Finally, there are many questions regarding the molecular mechanism of redox sensing by RegB and the specific involvement of upstream elements such as SenC and cytochrome cbb₃ oxidase in the redox control of RegB activity. There also remain many outstanding questions about the mechanism of transcription activation and repression by phosphorylated and dephosphorylated RegA, as well as the nature of the possible interactions of RegA with other transcription factors at target promoters. Hopefully, the decade to come will be as fruitful in unraveling the mysteries of the Reg regulon as was the decade that followed its discovery.

ACKNOWLEDGMENTS

 
We thank the numerous investigators who work on the RegB-RegA system for sending us recently published papers and communicating unpublished data.

RegB-RegA work from the Bauer laboratory is supported by grant GM 40941 from the National Institutes of Health.

FOOTNOTES

 
* Corresponding author. Mailing address: Department of Biology, Indiana University, Jordan Hall, Bloomington, IN 47405. Phone: (812) 855-6595. Fax: (812) 856-4178. E-mail: cbauer{at}bio.indiana.edu.

S.E., L.R.S., and D.L.S. contributed equally to this work and should be considered co-first authors.

REFERENCES

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