Search

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal "LytTR" DNA-binding domain. Here the RcoM(Bx)-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (~10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ~60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5'-TTnnnG-3') that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved "TTnnnG" motif residues and their triplet arrangement and were also used to determine an improved binding motif [5'-CnnC(C/A)(G/A)TTCAnG-3'] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM(Bx)-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoM(Bx)-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional "accumulating switch" that senses low but persistent CO levels.

Low levels of carbon monoxide inhibit the N(2)-dependent growth of Rhodospirillum rubrum unless the ∼100-residue CowN protein is expressed. Expression requires the CO-responsive regulator RcoM and is maximal in cells grown in the presence of CO and a poor nitrogen source, consistent with the role of CowN in N(2) fixation.

Genomic analysis suggested the existence of a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain. UV/visible-light spectral analyses of heterologously expressed, purified full-length proteins indicated that they contained a hexacoordinated b-type heme moiety that avidly binds CO and NO. Studies of protein variants strongly suggested that the PAS domain residues His74 and Met104 serve as the heme Fe(II) axial ligands, with displacement of Met104 upon binding of the gaseous effectors. Two RcoM (regulator of CO metabolism) homologs were shown to function in vivo as CO sensors capable of regulating an aerobic CO oxidation (cox) regulon. The genetic linkage of rcoM with both aerobic (cox) and anaerobic (coo) CO oxidation systems suggests that in different organisms RcoM proteins may control either regulon type.

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.

The CooA family of proteins are prokaryotic CO-sensing transcription factors that regulate the expression of genes involved in the utilization of CO as an energy source. They are homodimeric proteins that contain two hemes. Each monomer contains an N-terminal heme-binding domain and a C-terminal DNA-binding domain. Binding of CO to the heme leads to activation by a large reorientation of the DNA-binding domain such that the DNA-binding domain is in position for specific DNA recognition. The crystal structure of CooA from Rhodospirillum rubrum [RrCooA; Lanzilotta et al. (2000), Nature Struct. Biol. 7, 876-880] in the inactive CO-free off-state shows that the N-terminal Pro residue of monomer A coordinates the heme of monomer B and vice versa. It now appears that the CO replaces the Pro ligand and that this change is coupled to the activation process. However, precisely how the replacement of the Pro ligand by CO results in structural changes some 25 A from the CO-binding site remains unknown. Here, the structure of a CooA variant from the thermophilic bacterium Carboxydothermus hydrogenoformans (ChCooA) is reported in which one monomer is fully in the on-state. The N-terminal region that is displaced by CO binding is now positioned between the heme-binding and DNA-binding domains, which requires movement of the N-terminus by approximately 20 A and thus serves as a bridge between the two domains that helps to orient the DNA-binding domain in position for DNA binding.

The heme-containing transcriptional factor CooA regulates the expression of genes involved in the oxidation of carbon monoxide (CO) in the bacterium Rhodospirillum rubrum. CooA is both a redox sensor and a specific CO sensor, a combination of properties that is unique among heme proteins. Extensive biochemical and genetic analyses, interpreted in the context of a crystal structure of one form of the protein, have allowed the creation of hypotheses concerning the mechanism of CooA activation by CO as well as the basis for its CO specificity. The article details the data in support of these hypotheses and indicates future lines of research.

A unique feature of CooA, a heme-containing transcription factor, is that the N-terminal proline is the distal heme ligand in the ferrous state, and this ligand is displaced upon CO binding. To investigate the importance of Pro(2) in CO-dependent DNA binding, several CooA variants that alter N-terminal ligation were characterized. Electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectra of these variants provide the most definitive evidence that Pro(2) is the distal ligand in Fe(III) CooA. Furthermore, the functional and spectroscopic properties of these proteins depended on whether a weak ligand occupied the distal heme coordination site: for CooA variants in which distal coordination is disrupted, the DNA-binding affinities and Fe(II)-CO spectral properties showed an unexpected dependence on the order of CO addition and heme reduction. If N-terminal variant samples were incubated with CO before the heme was reduced, the proteins displayed DNA-binding affinities and Fe(II)-CO spectral characteristics similar to those of wild-type (WT) CooA. However, if the same samples were incubated with CO after the heme was reduced, the extent of functional and spectral similarity to WT CooA negatively correlated with the amount of high-spin heme present in the ferric state. From these data, it was inferred that the absence of a distal heme ligand in the ferric state prevents WT-like CO binding to the ferrous state, and it was hypothesized that correct CO binding is inhibited by the collapse of the distal heme pocket upon reduction. Together with the observation that L116H CooA, a variant in which His(116) replaces Pro(2) as the distal heme ligand, binds CO more slowly than WT CooA, these data indicate that the presence of a weak distal heme ligand, not specifically ligation by the N-terminal proline, is crucial for proper function. The role of Pro(2) in CooA is apparently to direct CO to bind on the distal side of heme and to help maintain the integrity of the distal heme pocket during the redox-mediated ligand switch.

The CO-specific heme-based sensor CooA regulates the ability of Rhodospirillum rubrum to grow on CO as an energy source. Only CO triggers the conformational change of CooA essential for the protein to function as a transcriptional activator. A structurally informed mutagenesis, followed by an in vivo screening method, allowed the isolation of a series of novel CooA variants that show very substantial response to imidazole. Compared with wild-type CooA, the ligand selectivity between imidazole and CO had been changed in some variants by roughly three orders of magnitude. Remarkably, different CooA variants also showed the ability to discriminate among imidazole derivatives, strongly implying a mechanism of precise interactions between the affected residues and the various ligands. Although wild-type CooA and imidazole-responsive CooA variants appear to recognize their respective ligands by fundamentally different mechanisms, several lines of evidence suggest that they respond by a similar C-helix repositioning that results in the rearrangement of the DNA-binding domains responsible for specific DNA contact. These results have implications for the molecular basis of both the imidazole responsiveness in the variants and the stringent CO specificity of wild-type CooA.

Carbon monoxide (CO) has long been known to have dramatic physiological effects on organisms ranging from bacteria to humans, but recently there have a number of suggestions that organisms might have specific sensors for CO. This article reviews the current evidence for a variety of proteins with demonstrated or potential CO-sensing ability. Particular emphasis is placed on the molecular description of CooA, a heme-containing CO sensor from Rhodospirillum rubrum, since its biological role as a CO sensor is clear and we have substantial insight into the basis of its sensing ability.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.

CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of R. rubrum. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.

CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum. CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism. To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays. Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO. The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact. The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket. The proximal Fe-His bond in Fe[II]CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain. A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.

CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium Rhodospirillum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO. As with other heme proteins, CooA is unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile. Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heme is able to bind CO. CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase. CooA is therefore a novel redox sensor as well as a specific CO sensor. CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time. The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors.

CooA is a carbon monoxide- (CO-) sensing homodimeric heme protein that activates the transcription of genes required for the anaerobic oxidation of CO to CO(2) in the phototrophic bacterium Rhodospirillum rubrum. In this study, we demonstrate that mutational alteration of the histidine residue (His(77)) that serves as a heme ligand in the Fe(II) form of CooA allows high-affinity binding of cyanide (K(d) approximately 0.4 mM) to the heme. In contrast, neither these same variants in the Fe(III) form nor wild-type CooA in either oxidation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyanide suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe(II) CooA without added effectors is also pH-dependent, suggesting that an ionizable amino acid has become solvent-accessible in the absence of His(77). Finally, we demonstrate that the transcriptional activity of H77Y CooA shows a small (1.4-fold) increase in the presence of cyanide, suggesting that the binding of cyanide to this variant promotes the active conformation of H77Y CooA.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with alpha subunits lacking their C-terminal domain (alpha-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that alpha-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not alpha-CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and alpha-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in alpha-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the alpha-CTD at class II promoters.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Resonance Raman spectra of the CO-responsive transcription factor CooA from Rhodospirillum rubrum provides evidence on the nature of heme ligation and its CO activation mechanism. The Fe(III) form gives standard low-spin heme spectrum, while the Fe(II) form is low spin for wild-type (WT) CooA and mixed spin for a CooA variant, H77Y, with an His77Tyr substitution. The Fe(II) porphyrin skeletal mode nu11 is at a value (1541 cm-1) indicative of a neutral donor ligand for the H77Y variant but is at an unusually depressed frequency (1529 cm-1) for the WT protein, indicating a strongly donating ligand. This ligand is proposed to be His77 imidazolate, formed by proton transfer to a nearby acceptor. The WT CO adduct has FeCO and CO stretching frequencies that indicate a neutral trans ligand and negative polarity in the CO binding pocket, while the CO adduct of His77Tyr has both 6- and 5-coordinate signals and a nonpolar CO environment. Photolysis of the WT CO adduct by the Raman laser produced a low-spin product at steady state, indicating fast recombination of the displaced ligand. These data suggest a novel YH- - -His- charge relay mechanism for CooA activation by CO. In this mechanism, His77 is reprotonated upon ligand displacement by CO; CO displaces either His77 or the trans ligand, X. The resulting charge on Y- may induce the protein conformation change required for site-selective DNA binding.

Biological sensing of small molecules such as NO, O2, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein's response to CO is proposed.

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2. This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.

In Rhodospirillum rubrum, CO induces the expression of at least two transcripts that encode an enzyme system for CO oxidation. This regulon is positively regulated by CooA, which is a member of the cAMP receptor protein family of transcriptional regulators. The transcriptional start site of one of the transcripts (cooFSCTJ) has been identified by primer extension. The ability of CooA to bind to this promoter in vitro was characterized with DNase I footprinting experiments using extracts of a CooA-overproducing strain. CooA- and CO-dependent protection was observed for a region with 2-fold symmetry (5'-TGTCA-N6-CGACA) that is highly similar to the consensus core motifs recognized by cAMP receptor protein/FNR family. In vivo analysis in a heterologous background indicates that CooA is sufficient for CO-dependent expression, implicating it as the likely CO sensor.

Induction of the CO-oxidizing system of the photosynthetic bacterium Rhodospirillum rubrum is regulated at the level of gene expression by the presence of CO. In this paper, we describe the identification of a gene that is required for CO-induced gene expression. An 11-kb deletion of the region adjacent to the previously characterized cooFSCTJ region resulted in a mutant unable to synthesize CO dehydrogenase in response to CO and unable to grow utilizing CO as an energy source. A 2.5-kb region that corresponded to a portion of the deleted region complemented this mutant for its CO regulation defect, restoring its ability to grow utilizing CO as an energy source. When the 2.5-kb region was sequenced, one open reading frame, designated cooA, predicted a product showing similarity to members of the cyclic AMP receptor protein (CRP) family of transcriptional regulators. The product, CooA, is 28% identical (51% similar) to CRP and 18% identical (45% similar) to FNR from Escherichia coli. The insertion of a drug resistance cassette into cooA resulted in a mutant that could not grow utilizing CO as an energy source. CooA contains a number of cysteine residues substituted at, or adjacent to, positions that correspond to residues that contact cyclic AMP in the crystal structure of CRP. A model based on this observation is proposed for the recognition of CO by Cooa. Adjacent to cooA are two genes, nadB and nadC, with predicted products similar to proteins in other bacteria that catalyze reactions in the de novo synthesis of NAD.(ABSTRACT TRUNCATED AT 250 WORDS)

Under dark, anaerobic conditions in the presence of sufficient nickel, Rhodospirillum rubrum grows with a doubling time of under 5 h by coupling the oxidation of CO to the reduction of H+ to H2. CO-dependent growth of R. rubrum UR294, bearing a kanamycin resistance cassette in cooC, depends on a medium nickel level ninefold higher than that required for optimal growth of coo+ strains.

Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.