ABC transporters associated with cytochrome c biogenesis.

It is generally agreed that cytochrome c biogenesis requires that the apocytochrome and heme be transported separately to their site of function and assembly. In bacteria, this is outside the cytoplasmic membrane, whereby the apocytochromes c use sec-dependent signals for their translocation. Two different hypotheses have recently emerged as to how heme is exported: one involves an helABCD-encoded ATP binding cassette (ABC) transporter complex and the second does not. The second hypothesis concludes that an (HelAB)2 heterodimeric ABC transporter does not transport heme but some other substrate for cytochrome c biogenesis. The evidence supporting each of these two hypotheses and the role of this ABC transporter is discussed.

Urea utilization in the phototrophic bacterium Rhodobacter capsulatus is regulated by the transcriptional activator NtrC.

The phototrophic nonsulfur purple bacterium Rhodobacter capsulatus can use urea as a sole source of nitrogen. Three transposon Tn5-induced mutations (Xan-9, Xan-10, and Xan-19), which led to a Ure(-) phenotype, were mapped to the ureF and ureC genes, whereas two other Tn5 insertions (Xan-20 and Xan-22) were located within the ntrC and ntrB genes, respectively. As in Klebsiella aerogenes and other bacteria, the genes encoding urease (ureABC) and the genes required for assembly of the nickel metallocenter (ureD and ureEFG) are clustered in R. capsulatus (ureDABC-orf136-ureEFG). No homologues of Orf136 were found in the databases, and mutational analysis demonstrated that orf136 is not essential for urease activity or growth on urea. Analysis of a ureDA-lacZ fusion showed that maximum expression of the ure genes occurred under nitrogen-limiting conditions (e.g., serine or urea as the sole nitrogen source), but ure gene expression was not substrate (urea) inducible. Expression of the ure genes was strictly dependent on NtrC, whereas sigma(54) was not essential for urease activity. Expression of the ure genes was lower (by a factor of 3.5) in the presence of ammonium than under nitrogen-limiting conditions, but significant transcription was also observed in the presence of ammonium, approximately 10-fold higher than in an ntrC mutant background. Thus, ure gene expression in the presence of ammonium also requires NtrC. Footprint analyses demonstrated binding of NtrC to tandem binding sites upstream of the ureD promoter. Phosphorylation of NtrC increased DNA binding by at least eightfold. Although urea is effectively used as a nitrogen source in an NtrC-dependent manner, nitrogenase activity was not repressed by urea.

Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model.

Unlike other cytochromes, c-type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c-type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild-type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N-terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin-like protein. Both the B. pertussis dipZ (i. e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol-reducing compounds, dithiothreitol and 2-mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435-amino-acid periplasmic region.

Oxidation-reduction properties of disulfide-containing proteins of the Rhodobacter capsulatus cytochrome c biogenesis system.

Oxidation-reduction titrations for the active-site disulfide/dithiol couples of the helX- and ccl2-encoded proteins involved in cytochrome c biogenesis in the purple non-sulfur bacterium Rhodobacter capsulatus have been carried out. The R. capsulatus HelX and Ccl2 proteins are predicted to function as part of a dithiol/disulfide cascade that reduces a disulfide on the apocytochromes c so that two cysteine thiols are available to form thioether linkages between the heme prosthetic group and the protein. Oxidation-reduction midpoint potential (E(m)) values, at pH 7.0, of -300 +/- 10 and -210 +/- 10 mV were measured for the HelX and Ccl2 (a soluble, truncated form of Ccl2) R. capsulatus proteins, respectively. Titrations of the disulfide/dithiol couple of a peptide designed to serve as a model for R. capsulatus apocytochrome c(2) have also been carried out, and an E(m) value of -170 +/- 10 mV was measured for the model peptide at pH 7.0. E(m) versus pH plots for HelX, Ccl2, and the apocytochrome c(2) model peptide were all linear over the pH range from 5.0 to 8.0, with the -59 mV/pH unit slope expected for a reaction in which two protons are taken up for each disulfide that is reduced. These results provide thermodynamic support for the proposal that HelX reduces Ccl2 and that reduced Ccl2, in turn, serves as the reductant for the production of the two thiols of the CysXxxYyyCysHis heme-binding motif of the apocytochromes.

In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus.

It has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen. During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions. We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light-harvesting II apoproteins) and puf (reaction centre and light-harvesting I apoproteins) using purified transcription factors and RNA polymerase from Rhodobacter capsulatus. Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two-component regulators RegA/RegB, which are activators of puc and puf. These regulators are distinct from those that mediate oxygen control in enteric bacteria. Our in vitro studies show that these purified regulators directly control the expression of the housekeeping RNA polymerase at these promoters. High-level basal expression of the bch promoter is shown to be repressed by CrtJ. The puc promoter is activated by the RegB-phosphorylated RegA protein and additionally repressed by CrtJ. At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site. In contrast to what has been suggested previously, the RegA-activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase. We also discuss evidence that RegA approximately P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein-protein interaction.

Transmembrane heme delivery systems.

Heme proteins play pivotal roles in a wealth of biological processes. Despite this, the molecular mechanisms by which heme traverses bilayer membranes for use in biosynthetic reactions are unknown. The biosynthesis of c-type cytochromes requires that heme is transported to the bacterial periplasm or mitochondrial intermembrane space where it is covalently ligated to two reduced cysteinyl residues of the apocytochrome. Results herein suggest that a family of integral membrane proteins in prokaryotes, protozoans, and plants act as transmembrane heme delivery systems for the biogenesis of c-type cytochromes. The complete topology of a representative from each of the three subfamilies was experimentally determined. Key histidinyl residues and a conserved tryptophan-rich region (designated the WWD domain) are positioned at the site of cytochrome c assembly for all three subfamilies. These histidinyl residues were shown to be essential for function in one of the subfamilies, an ABC transporter encoded by helABCD. We believe that a directed heme delivery pathway is vital for the synthesis of cytochromes c, whereby heme iron is protected from oxidation via ligation to histidinyl residues within the delivery proteins.

Translational activation by an NtrC enhancer-binding protein.

The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-binding protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor. The nifR3-ntrB-ntrC operon in R. capsulatus codes for the nitrogen-sensing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes. Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported. The nifR3-ntrB-ntrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and ntrC mutants under nitrogen-limited or nitrogen-sufficient conditions. LacZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain constant, production of NifR3 is at least ten to 40-fold reduced in NtrC- strains. NifR3 is increased at least fivefold upon nitrogen limitation whereas NtrC production is constitutive. Surprisingly, the purified NtrC protein binds cooperatively to the nifR3 promoter region in vitro at two sets of tandem binding sites centered at +1 and -81 nucleotides relative to the transcriptional start site. Deletion analysis demonstrates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo , but are not necessary for nifR3 transcription. These experiments indicate that NtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA. This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA and anfA) in R. capsulatus which are transcriptionally activated by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site.

A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase.

A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping sigma70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called sigma54 or sigmaN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/sigma54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/sigma70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed.

Comparison of the bacterial HelA protein to the F508 region of the cystic fibrosis transmembrane regulator.

The HelA protein of Rhodobacter capsulatus is the ATP-binding-cassette subunit of an exporter complex required for cytochrome c biogenesis. By primary sequence comparisons the F88 residue of HelA is similar to the F508 residue of the cystic fibrosis transmembrane regulator (CFTR) protein. Previous studies have established that CFTR F508delta or F508R proteins are defective but F508C is functional. Our results demonstrate that the HelA F88 mutants functionally mimic the phenotypes of known CFTR F508 mutants. The phenotypes of additional HelA mutants and the in vivo steady-state levels of these proteins are also reported.

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.

Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase. In vitro transcription of photosynthesis and other genes.

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma70 factor (R. capsulatus RNAP/sigma70) was purified and characterized using two classical sigma70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli sigma70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c2 (cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma70 indicated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.

Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments.

A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies.

The c-type cytochromes are distinguished from other heme proteins by the covalent ligation of two heme vinyl groups to two cysteine residues on the apoprotein (at a CXXCH domain). The present study was undertaken to elucidate the roles and topological locations of two of the proteins necessary for cytochrome c biogenesis, the HelX and Ccl2 proteins in the Gram-negative bacteria Rhodobacter capsulatus. From their primary sequence, each of these proteins has a CXXC motif that could be involved in the reduction of the cysteine residues of the apocytochromes c, a prerequisite for covalent ligation to the heme. Results of site-directed mutagenesis of HelX and Ccl2 demonstrate that each cysteine residue is required for the in vivo function of the protein. We demonstrate that the native HelX in R. capsulatus is tethered to the cytoplasmic membrane via its uncleaved signal sequence. Ccl2 is tethered by a single transmembrane domain present in the C terminus with the N-terminal two-thirds of the protein in the periplasm. Thus, both CXXC motifs are exposed to the periplasm. The complete HelX protein and the soluble N-terminal portion of Ccl2 (called Ccl2*) were overproduced and purified from periplasmic fractions. The Ccl2* signal sequence is efficiently processed. In vitro studies with these purified proteins indicate that although neither can reduce insulin, HelX can reduce the Ccl2 cysteine residues and the Ccl2 cysteine residues are oxidized by an apocytochrome c peptide containing the CXXCH domain. Revertants of an helX deletion mutant were isolated that regain the ability to make c-type cytochromes (and thus grow photosynthetically); some of these suppressor strains are enhanced for photosynthetic growth by the addition of thio-reducing agents. In contrast, revertants of a ccl2 deletion strain could not be isolated under any condition. These results suggest that the HelX and Ccl2 proteins form a thioreduction pathway (HelX-->Ccl2-->apocytochrome c) whereby Ccl2 function may be highly specific for apocytochromes c while HelX may act as a more general reductant of proteins with vicinal cysteines.

Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes.

Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology.

Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production.

Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis.

The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters.

Use of heme reporters for studies of cytochrome biosynthesis and heme transport.

Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes. The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm. To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E. coli. By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant. Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm. Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells. Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space. Such an imbalance could lead to defects in the synthesis of heme-liganded proteins. The heme reporters were also used to analyze strains of E. coli with a defect in genes encoding homologs of a different ABC transporter (helABC). The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R. G. Kranz, J. Bacteriol. 171:456-464, 1989; D. L. Beckman, D. R. Trawick, and R. G. Kranz, Genes Dev. 6:268-283, 1992). This locus was shown to be essential in E. coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis. Consistent with these and previous results, it is proposed that the HelABC transporter is specifically involved in heme export for ligation (hel). This class of periplasmic cytochromes is proposed to require heme liganding before undergoing correct folding.

The temperature-sensitive growth and survival phenotypes of Escherichia coli cydDC and cydAB strains are due to deficiencies in cytochrome bd and are corrected by exogenous catalase and reducing agents.

The cydDC operon of Escherichia coli encodes an ATP-dependent transporter of unknown function that is required for cytochrome bd synthesis. Strains containing defects in either the cydD or cydC gene also demonstrate hypersensitivity to growth at high temperatures and the inability to exit the stationary phase at 37 degrees C. We wished to determine what is responsible for these hypersensitive phenotypes and whether they are due to a lack of the CydDC proteins or a defect of the cytochrome bd encoded by the cydAB genes. Using both K-12- and B-type strains of E. coli, we have compared the phenotypes of isogenic cydAB mutants and cydC mutants. In both K-12- and B-type backgrounds, the hypersensitive phenotypes are due to defects of cytochrome bd activity and not defects of the cydDC genes. We also found that the temperature-sensitive growth phenotypes can be suppressed by exogenous reducing agents, such as glutathione and cysteine. Strikingly, even the enzymes catalase and superoxide dismutase, when added exogenously, can correct the temperature-sensitive and stationary phase arrest phenotypes. We propose that the temperature sensitive growth phenotypes are due to a buildup of diffusible oxygen radicals brought on by the absence of cytochrome bd.

In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system.

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.