Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1

Abstract

Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.

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

“…putida strain DS1, which utilized DMS or DMSO as a sulfur source, was recently isolated (Endoh et al, 2003). When strain DS1 grew on DMS as a sulfur source, DMSO and dimethyl sulfone (DMSO 2 ) accumulated in the culture.…”

“…DMS-utilization-defective mutants of strain DS1 were obtained by Tn5 mutagenesis. One mutant, Dfi175, no longer utilized DMS, DMSO, DMSO 2 and MSA as sulfur sources, and had a deficiency in the ssuEADCBF operon, which encodes an ABC-transporter (SsuABC), a two-component sulfonate sulfonatase system (SsuED) and a small protein SsuF (Endoh et al, 2003). The ssuEADCBF operon has also been reported to be essential in P. putida S-313 for utilization of organosulfur compounds, including sulfonates, sulfate esters and methionine (Kahnert et al, 2000).…”

“…However, the accumulation of DMSO and DMSO 2 was repressed in the presence of sulfate. This suggests that enzymes involved in DMS oxidation are regulated by SSR (Endoh et al, 2003). DMS-utilization-defective mutants of strain DS1 were obtained by Tn5 mutagenesis.…”

“…DNA fragments were extracted from the agarose gel by using Concert Rapid Gel Extraction Systems (Invitrogen) according to the manufacturer's instructions. We used a modified cosmid vector, SuperCos1DNEO (Ap r Km s ; Endoh et al, 2003), for construction of cosmid library and Tn5 rescue from mutants. Southern, Northern and colony hybridization analyses were carried out by standard methods (Sambrook et al, 1989), using digoxigenin (DIG)-labelled probes synthesized with a DIG DNA labelling and detection system (Roche Diagnostics) or 32 Plabelled probes synthesized with a Megaprime DNA labelling system (Amersham Pharmacia Biotech) and [a- Amersham Pharmacia Biotech).…”

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…Transfers of pCAR1 to P. resinovorans CA10dm4 and P. putida KT2440 were detected experimentally at frequencies of 3 Â 10 À1 and 3 Â 10 À3 per donor cell, 57) suggesting that pCAR1 is in fact a self-transmissible plasmid. Mating experiments showed that pCAR1 can transfer from P. putida HS01 (P. putida DS1 85) harboring pCAR1) to P. chlororaphis, P. fluorescens, and P. stutzeri, although no transfer of pCAR1 from P. resinovorans CA10 to them was detected. 86) Hence we concluded that the recipient range of pCAR1 was affected largely by the donor cell.…”