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The σ 54 ‐dependent transcriptional activator SfnR regulates the expression of the Pseudomonas putida sfnFG operon responsible for dimethyl sulphone utilization
Author(s) -
Endoh Takayuki,
Habe Hiroshi,
Nojiri Hideaki,
Yamane Hisakazu,
Omori Toshio
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04431.x
Subject(s) - operon , pseudomonas putida , biology , mutant , transposon mutagenesis , biochemistry , mutagenesis , gene , complementation , monooxygenase , lac operon , transposable element , microbiology and biotechnology , plasmid , enzyme , cytochrome p450
Summary Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO 2 ), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a σ 54 ‐dependent transcriptional regulator is essential for DMSO 2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO 2 ‐utilization‐defective phenotype) using mini‐Tn 5 , which contains two outward‐facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO 2 . The DMSO 2 ‐positive mutant carried a mini‐Tn 5 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 FMNH 2 ‐dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO 2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH 2 ‐dependent DMSO 2 monooxygenase that converts DMSO 2 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.