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

Abstract

Rhodobacter sphaeroides is a gram-negative purple nonsulfur bacterium belonging to the alpha-3 subdivision of the Proteobacteria (49). This organism is capable of a wide range of metabolic lifestyles, exhibiting growth chemoautotrophically, photoautotrophically, photoheterotrophically, chemoheterotrophically, and diazotrophically (reviewed in reference 24). The ability of purple nonsulfur bacteria, as well as other bacterial species, to utilize dimethyl sulfoxide (DMSO) and trimethylamine N-oxide (TMAO) as terminal electron acceptors in the absence of oxygen for chemoheterotrophic growth has been well documented (12,23,52). Both DMSO and TMAO are commonly occurring compounds in nature and play roles in sulfur and nitrogen cycling (1).Under anoxic conditions in the presence of DMSO or TMAO and in the absence of light, R. sphaeroides can grow via a periplasmic DMSO reductase (DMSOR), which serves as the terminal electron acceptor in a cascade in which electrons are transferred from quinol to DMSO or TMAO (24). This process does not require the cytochrome bc 1 complex, and electrons are transferred from quinol to DMSOR via a membranebound b-type cytochrome and a periplasmic c-type cytochrome (25). DMSOR from both R. sphaeroides and the closely related bacterium R. capsulatus have been crystallized and shown to contain the molybdopterin cofactor (Moco) as the sole prosthetic group (for a review, see reference 21).Studies on the genetics of DMSO reduction in both R. sphaeroides and R. capsulatus have been limited mainly to several reports which describe the sequence of the gene encoding DMSOR (2, 22, 37, 51). The dmsA or dorA gene product has extensive homology to other molybdoenzymes such as biotin sulfoxide reductase and TMAO reductase (50). Recently, the sequences of two genes upstream of the dmsA gene of R. sphaeroides f. sp. denitrificans were reported (45). The dmsC and dmsB gene products were shown to be homologous to the torC and torD gene products, which encode a c-type cytochrome and a membrane protein, respectively, and it was suggested that the dmsCBA genes form a transcriptional unit, similar to the torCDA genes in E. coli (27). Studies from our laboratory revealed that R. sphaeroides 2.4.1 T possesses two different circular chromosomes, of ϳ3.0 Mbp (CI) and ϳ0.9 Mbp (CII) (42,43). A number of genes have been shown to be duplicated between CI and CII (5). In contrast, we have previously shown by Tn5 mutagenesis that some pathways, e.g., p-aminobenzoic acid, uracil, histidine, and thymine biosynthesis, are partitioned rather than duplicated between CI and CII (4). These results suggested that CII is an essential genomic element, having unique as well as shared and duplicated functions.Recently, we used a low-redundancy sequencing strategy for analysis of the genetic content of CII (5). We analyzed ϳ300 kb of unique DNA which identified approximately 200 putative open reading frames (ORFs) representing a wide variety of functions, e.g., amino acid biosynthesis, nutrient transporters, redox-active systems, ...

TorD, A Cytoplasmic Chaperone That Interacts with the Unfolded Trimethylamine N-Oxide Reductase Enzyme (TorA) in Escherichia coli

“…Alternatively, another chaperone, which could be either a general molecular chaperone or a specific chaperone (for example, EcYcac), takes over the folding of TorA. Surprisingly, Moncey et al (28) have recently proposed that R. sphaeroides DmsB protein possesses an essential role in the TMAO respiratory pathway. Experiments are in progress to confirm that the TorD homologues play the same role toward their cognate enzyme as that of the E. coli TorD protein.…”

Electron Transfer and Binding of the c-Type Cytochrome TorC to the Trimethylamine N-Oxide Reductase in Escherichia coli

“…In TMAO and Me 2 SO reduction systems, pentahemic cytochromes proved to be essential for the electron transfer and thus appeared to be the intermediate components between the membrane quinone pool and the terminal reductase (6,22). Since it has been shown in E. coli that TMAO reduction involves menaquinones (23), TorC should receive the electrons from the menaquinone pool (EЈ 0(menaquinol/menaquinone) ϭ Ϫ74 mV) and transfer them to the active site of TorA, where TMAO reduction (EЈ 0(TMAO/TMA) ϭ ϩ130 mV) occurs.…”

The role of dor gene products in controlling the P2 promoter of the cytochrome c 2 gene, cycA, in Rhodobacter sphaeroides

“…The 215 bp dorCBA promoter fragment was generated by PCR using the DorC-1 (59-GCGAGGA-AGCTTGCGGCCGGTTGGACCTAATGC-39) and DorC-2 (59-CCC-GGATCCGCAGGAATGCGGCGGGCG-39) primers, using pNMT16 as a template (Mouncey et al, 1997). The PCR product was purified from an agarose gel using a Qiagen Gel Extraction Kit.…”

“…One possibility is that this effect depends on the function of a protein that requires DorR for its synthesis or activity: for instance, one of the dorCBA gene products (Mouncey & Kaplan, 1998). To determine if any dorCBA gene products are required for the DMSOdependent decrease in expression from cycA P2 : : lacZ under photosynthetic conditions, we tested expression of this promoter in cells containing an insertion in dorC that has been shown to be polar onto dorBA (Mouncey et al, 1997). In the DorC 2 strain, the presence of DMSO did not decrease activity of cycA P2 under photosynthetic conditions (Fig.…”

“…The properties of DorR 2 mutants predict that DorR is required for activation of the dorCBA operon, which encodes a c-type cytochrome (DorC), a putative membrane protein of unknown function (DorB), and the DMSO reductase enzyme (DorA) (Mouncey & Kaplan, 1998;Mouncey et al, 1997). The~100 bp of DNA between the divergent dorCBA and dorR open reading frames contains four proposed DorR binding sites (Mouncey & Kaplan, 1998) that share sequence similarity with elements bound by the related response regulator, TorR, in E. coli (Simon et al, 1994).…”