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, ...