Respiratory pathways of Rhodobacter sphaeroides 2.4.1(T): identification and characterization of genes encoding quinol oxidases.

Rhodobacter sphaeroides 2.4.1(T) respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases. Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized. The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa. No phenotype was observed for mutations in either oxidase in the wild-type background. A strain containing a qxtA mutation in a cytochrome bc(1) complex mutant background was unable to grow aerobically. No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions. These are the first molecular studies to characterize the quinol oxidases of R. sphaeroides 2.4.1(T).

RsGDB, the Rhodobacter sphaeroides Genome Database.

This report provides a summary of the sequencing project of the small chromosome (CII) of Rhodobacter sphaeroides 2.4.1(T),and introduces the first version of the genome database of this bacterium. The database organizes and describes diverse sets of biological information. The main role of the R.sphaeroides genome database (RsGDB) is to provide public access to the collected genomic information for R.sphaeroides via the World-Wide Web at http://utmmg.med.uth.tmc.edu/sphaeroides. The database allows the user access to hundreds of low redundancy R.sphaeroides sequences for further database searching, a summary of our current search results, and other allied information pertaining to this bacterium.

Redox-dependent gene regulation in Rhodobacter sphaeroides 2.4.1(T): effects on dimethyl sulfoxide reductase (dor) gene expression.

The ability of Rhodobacter sphaeroides 2.4.1(T) to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus. Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and beta-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R. sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.

Cascade regulation of dimethyl sulfoxide reductase (dor) gene expression in the facultative phototroph Rhodobacter sphaeroides 2.4.1T.

Under anaerobic-dark growth conditions, in the presence of the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), Rhodobacter sphaeroides 2.4.1(T) respires anaerobically using the molybdoenzyme DMSO reductase (DMSOR). Genes encoding DMSOR and associated proteins are encoded by genes of the dor locus. Previously, we demonstrated that the expression of DMSOR is regulated by both the oxygen status of the cell via the FnrL protein and by the presence of DMSO or TMAO, presumably through the DorS-DorR two-component sensor-regulator system. Here we further investigate expression of the dor genes through the use of transcriptional lacZ fusions to the dorS, dorR, and dorC promoters. The expression of dorC::lacZ was strongly induced by the absence of oxygen and presence of DMSO. In accordance with our previous findings of DMSOR activity, dorC::lacZ expression was reduced by up to one-third when cells were grown photosynthetically in the presence of DMSO with medium or high light, compared to the expression observed after anaerobic-dark growth. The induction of dorC::lacZ expression in the presence of DMSO was dependent on the DorS and DorR proteins. Expression of the dorS and dorR genes was also induced in the absence of oxygen. In an FnrL mutant, dorS::lacZ expression was not induced when oxygen tensions in the media were lowered, in contrast to what occurred in the wild-type strain. The expression of dorS::lacZ and dorR::lacZ was dependent on the DorS and DorR proteins themselves, suggesting the importance of autoregulation. These results demonstrate a cascade regulation of dor gene expression, where the expression of the regulatory proteins DorS and DorR governs the downstream regulation of the dorCBA operon encoding the structural proteins of DMSOR.

Oxygen regulation of the ccoN gene encoding a component of the cbb3 oxidase in Rhodobacter sphaeroides 2.4.1T: involvement of the FnrL protein.

The ccoNOQP gene cluster of Rhodobacter sphaeroides 2.4.1T encodes a cbb3 cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration. The beta-galactosidase activity of a ccoN::lacZ transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions. Maximal ccoN::lacZ expression was observed when the oxygen concentration was lowered to 2%. In an FnrL mutant, ccoN::lacZ expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding the cbb3 oxidase.

Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II.

Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.

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.

The modE gene product mediates molybdenum-dependent expression of genes for the high-affinity molybdate transporter and modG in Azotobacter vinelandii.

The Azotobacter vinelandii mod locus, which is involved in high-affinity molybdate transport and the early event in Mo metabolism, consists of two divergently transcribed operons, modG and modEABC. modA, modB and modC encode the components of the high-affinity molybdate transporter, and modG encodes a Mo-binding protein. High concentrations of Mo repressed transcription of both operons. The modEABC operon was also repressed by tungstate and to a lesser extent by vanadate. modE, the first gene in the modEABC operon, controlled the Mo-dependent transcription of both operons. It was not involved in the metal regulation of alternative nitrogenase gene expression. Although a modE mutant constitutively expressed genes encoding the molybdate transporter, it had a reduced rate of Mo accumulation.

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.

DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.