The sigma54-dependent transcriptional activator SfnR regulates the expression of the Pseudomonas putida sfnFG operon responsible for dimethyl sulphone utilization.
Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO2), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a sigma54-dependent transcriptional regulator is essential for DMSO2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO2-utilization-defective phenotype) using mini-Tn5, which contains two outward-facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO2. The DMSO2-positive mutant carried a mini-Tn5 insertion in the intergenic region between two opposite-facing operons, sfnAB and sfnFG. Both sfnA and sfnB products were similar to acyl-CoA dehydrogenase family proteins, whereas sfnF and sfnG encoded a putative NADH-dependent FMN reductase (SfnF) and an FMNH2-dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH2-dependent DMSO2 monooxygenase that converts DMSO2 to MSA. It was revealed that the expression of the sfnFG operon is directly activated by the binding of SfnR at its upstream region. Site-directed mutagenesis of the SfnR binding sequences allowed us to define a potential recognition sequence for SfnR. These results provided insight into regulation of sulphate starvation-induced genes in bacteria.
Endoh
Takayuki
T
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan.
Habe
Hiroshi
H
Nojiri
Hideaki
H
Yamane
Hisakazu
H
Omori
Toshio
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004121:Dimethyl Sulfoxide; D015964:Gene Expression Regulation, Bacterial; D008698:Mesylates / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D051877:RNA Polymerase Sigma 54; D017422:Sequence Analysis, DNA; D012808:Sigma Factor / Q000378:metabolism*; D013450:Sulfones / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D008698:Mesylates; D012808:Sigma Factor; D013450:Sulfones; D015534:Trans-Activators; C045880:methanesulfonic acid; C025910:dimethyl sulfone; D006899:Mixed Function Oxygenases; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D004121:Dimethyl Sulfoxide