A CysB-regulated and σ54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1

Abstract

Pseudomonas putida strain DS1 utilizes dimethyl sulfide (DMS) as a sulfur source, and desulfurizes it via dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ) and methanesulfonate (MSA). Its Tn5 mutant, Dfi74J, no longer utilized DMS, DMSO and DMSO 2 , but could oxidize DMS to DMSO 2 , suggesting that the conversion of DMSO 2 to MSA was interrupted in the mutant. Sequencing of the Tn5 flanking region of Dfi74J demonstrated that a gene, sfnR (designated for dimethyl sulfone utilization), encoding a transcriptional regulator containing an ATP-dependent s 54 -association domain and a DNA-binding domain, was disrupted. sfnR is part of an operon with two other genes, sfnE and sfnC, located immediately upstream of sfnR and in the same orientation. The genes encode NADH-dependent FMN reductase (SfnE) and FMNH 2 -dependent monooxygenase (SfnC). Complementation of Dfi74J with an sfnR-expressing plasmid led to restoration of its growth on DMS, DMSO and DMSO 2 . An rpoN-defective mutant of strain DS1, which lacks the s 54 factor, grew on MSA, but not on DMS, DMSO and DMSO 2 , indicating that SfnR controls expression of gene(s) involved in DMSO 2 metabolism by interaction with s 54 -RNA polymerase. Northern hybridization and a reporter gene assay with an sfn-lacZ transcriptional fusion elucidated that expression of the sfnECR operon was induced under sulfate limitation and was dependent on a LysR-type transcriptional regulator, CysB. This is believed to be the first report that a s 54 -dependent transcriptional regulator induced under sulfate limitation is involved in sulfur assimilation.

Transcription Factors CysB and SfnR Constitute the Hierarchical Regulatory System for the Sulfate Starvation Response inPseudomonas putida

“…Because we had found a 54 -dependent promoter-like sequence upstream of the sfnE translation start point, we first believed that the expression of sfnECR was autoregulated by SfnR. However, in our previous study, the expression of sfnECR was observed in an sfnR-defective mutant (Dfi74J) (9). Therefore, we postulated that the expression of sfnECR is controlled by another 54 -dependent transcriptional regulator or that additional promoters exist in the upstream region of sfnECR.…”

“…Many 54 -dependent transcriptional regulators share a common domain architecture that includes three domains: a C-terminal DNA-binding domain, a central module for ATPase activity and interaction with 54 -RNA polymerase, and an N-terminal regulatory domain (37). However, as reported previously, SfnR lacks an N-terminal regulatory domain (8,9); therefore, it is of interest in this context to examine how sulfate is sensed and represses the expression of sfnFG. Several studies have reported on 54 -dependent transcriptional regulators lacking an N-terminal regulatory domain like SfnR.…”

“…Each amplified fragment was cloned into a pT7Blue (R) vector (Novagen), and the nucleotide sequences of each reporter plasmid were confirmed. Clones were digested with both HindIII and EcoRI (sites incorporated in the primers) (see Table S1 in the supplemental material), and the fragments were then cloned between the HindIII and EcoRI sites of pMElacZ (9).…”

“…More interestingly, SfnR lacks an N-terminal regulatory domain, unlike many other 54 -dependent transcriptional regulators (8,9). Therefore, determining the role that SfnR plays in the sulfate starvation response would be of great interest.…”

“…Expression of the sfnFG operon is directly regulated by a novel 54 -dependent transcriptional regulator, SfnR, which is encoded within the sfnECR operon (Fig. 1B) (8,9). SfnR also activates the expression of the sfnAB operon, and sfnA is suggested to be involved in the use of methanethiol as a sulfur source (11).…”