Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

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.

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.

SummaryPseudomonas 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 s s s s 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 miniTn 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. Sitedirected 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.