Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Toshio Omori
aseigyo{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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The DDBJ/EMBL/GenBank accession number for the sfnECR operon reported in this paper is AB091764.
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INTRODUCTION |
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A variety of bacteria in aquatic sediments and soil have been reported to utilize DMS and/or its related compounds including DMSO and methanethiol as a carbon, energy or sulfur source (de Bont et al., 1981; Smith & Kelly, 1988
; Omori et al., 1995
; Horinouchi et al., 1997
; Fuse et al., 2000
; Lomans et al., 2002
). Thus, it has been proposed that bacterial conversion of such organosulfur compounds is involved in the global sulfur cycle.
Several enteric and soil bacteria are able to utilize a variety of organosulfur compounds including taurine, MSA, alkanesulfonates and/or alkanesulfate esters as sulfur sources (Roberts et al., 1955; Uria-Nickelsen et al., 1993
; van der Ploeg et al., 1996
, 1998
, 1999
; Hummerjohann et al., 1998
; Vermeij et al., 1999
; Kertesz, 1999
). Recently, the genes involved in utilization of such organosulfur compounds in Escherichia coli, Bacillus subtilis, Pseudomonas putida and P. aeruginosa have been reported. The tauABCD and ssuEADCB operons of E. coli are required for taurine and alkanesulfonate utilization, respectively (van der Ploeg et al., 1996
, 1999
). The expression of tau and ssu operons is repressed in the presence of preferred sulfur sources such as inorganic sulfate, thiosulfate or cysteine, but induced in their absence (van der Ploeg et al., 1997
, 1999
). This phenomenon is called the sulfate starvation response (SSR; Quadroni et al., 1996
), and is controlled by two LysR-type transcriptional regulators, CysB and Cbl, in E. coli (van der Ploeg et al., 1997
, 1999
; Bykowski et al., 2002
). Also, in P. aeruginosa and P. putida S-313, induction of a set of proteins under sulfate starvation was observed (Quadroni et al., 1999
; Kahnert et al., 2000
). These sulfate-starvation-induced (SSI) proteins enable bacterial cells to adapt to sulfate limitation, e.g. they include enzymes for scavenging sulfur from suboptimal concentrations of organosulfur compounds. Although CysB plays an essential role in organosulfur utilization in P. aeruginosa (Kertesz et al., 1999
; Hummerjohann et al., 2000
), there are few reports concerning transcriptional regulation of SSI genes in Pseudomonas sp.
P. 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 (DMSO2) accumulated in the culture. However, the accumulation of DMSO and DMSO2 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. One mutant, Dfi175, no longer utilized DMS, DMSO, DMSO2 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
). Although the DMS metabolic pathway of P. putida strain DS1 was proposed as shown in Fig. 1
, enzymes or proteins involved in the conversion of DMS to MSA remained unclear.
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METHODS |
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DNA sequence analysis.
DNA sequences were analysed by the chain-termination method with a Li-Cor model 4200L-2 Auto-DNA Sequencer and Base ImagIR Data Collection Software 4.0 (Li-Cor) according to the manufacturer's instructions. Homology searching was performed using the SWISS-PROT amino acid sequence database or the DDBJ/EMBL/GenBank DNA databases with the BLAST program (Altschul et al., 1997).
Plasmid construction.
The rpoN gene was amplified from total DNA of strain DS1 by PCR (98 °C for 5 min; followed by 25 cycles of 98 °C for 1 min, 55 °C for 5 s, 74 °C for 2·5 min; then by 74 °C for 5 min) with a set of primers, Pp-rpoN-FW and Pp-rpoN-RV (Table 1) designed based on the 5'- and 3'-terminal ends, respectively, of rpoN from P. putida strains. A 1·5 kb amplified product was digested with EcoRI and ligated into pBluescript II SK(-), to produce pBSrpoN (Table 1
), and the insert was then sequenced to confirm that rpoN was encoded. Finally, a SalISphI internal region of the insert was subcloned into pUC19 to give pUC
rpoN (Table 1
), and the Kmr gene cassette (kan) from pTkm (Yoshida et al., 2003
) was then inserted into the ScaI site in the Apr gene of pUC
rpoN. The resultant plasmid, pUC
rpoN-Km, was used for disruption of rpoN in P. putida strain DS1.
The internal region of cysB was amplified from total DNA of strain DS1 by PCR (98 °C for 5 min; followed by 25 cycles of 98 °C for 1 min, 55 °C for 5 s, 74 °C for 1·5 min; then by 74 °C for 3 min) with a set of primers, Pp-cysB-int-F and Pp-cysB-int-R (Table 1) designed based on the internal sequence of cysB from P. aeruginosa strain PAO1. The resultant 865 bp product amplified was digested with EcoRI and ligated into pBluescriptII SK(-) to give pBS
cysB. The insert then was sequenced to confirm that it was the internal region of cysB. Finally, kan was ligated into the ScaI site in the Apr gene of pBS
cysB. The resultant plasmid, pBS
cysB-Km, was used for disruption of cysB of strain DS1.
For reporter gene assay, a broad-host-range reporter plasmid, pMElacZ, was constructed. The lacZ gene was amplified from pRS551 (Simons et al., 1987) by PCR (98 °C for 5 min; followed by 25 cycles of 98 °C for 1 min, 60 °C for 15 s, 74 °C for 5 min; then by 74 °C for 7 min) with lac-hindIII-F and lac-sfiI-R primers (Table 1
). A 245 bp HindIIISfiI fragment of pME4510 (Rist & Ketesz, 1998
) containing lacZ
was replaced by the HindIII-SfiI-digested lacZ gene, to give pMElacZ. A 1·9 kb EcoRVHindIII fragment of pBSsfn02 containing sfnE and its promoter region was cloned into SmaI-HindIII-digested pMElacZ, to produce pMEsfn-lacZ (Fig. 2
). pMEsfn-lacZ was used for assay of sfn promoter activity.
Measurement of growth characteristics.
Pseudomonas strains were grown on 1 ml SFMM with 1 mM cysteine (for the cysB mutant), or on 1 ml SF-M9 glucose medium with 1 mM Na2SO4 (for the rpoN mutant) at 30 °C with reciprocal shaking (300 strokes min-1). Cells were harvested by centrifugation (8000 r.p.m., 5 min) and washed three times by suspending into 1 ml of the respective sulfur-free medium. The washed cells were resuspended into 1 ml of the respective sulfur-free medium, and then 10 µl aliquots of the resultant cell suspension were inoculated into 1 ml of the respective sulfur-free medium containing an appropriate sulfur source. After appropriate intervals, the OD550 was measured using a spectrophotometer (model DU-7400; Beckman).
RNA preparation.
Total RNA was extracted using an RNeasy Mini kit combined with a QIAshredder and RNase-Free DNase set from Qiagen according to the manufacturer's instructions. The isolated total RNA was subjected to Northern hybridization according to Sambrook et al. (1989).
RT-PCR.
RT-PCR was carried out by using TaKaRa One Step RNA PCR Kit (AMV) (Takara Shuzo). The reaction mixture (50 µl) contained 5 µl 10-fold One Step RNA PCR Buffer, 50 µM MgCl2, 10 µM dNTP, 40 U RNase inhibitor, 5 U AMV RTase XL, 5 U AMV-Optimized Taq, 20 µM each of rt-sfnC-FW and rt-sfnR-RV (Table 1), and 1 µg total RNA prepared as described above. The temperature was raised to 50 °C for 30 min, then to 94 °C for 2 min followed by 25 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and was finally ramped down to 4 °C by a GeneAmp PCR System 9700.
Measurement of sfn promoter activity.
Strains DS1(pMEsfn-lacZ) and CYSB(pMEsfn-lacZ) were grown at 30 °C on a reciprocal shaker in sets of 20 tubes of SF-M9 glucose medium (1 ml) containing 1 mM cysteine. The cells were harvested and washed by suspending in SF-M9 (1 ml), and then resuspended in SF-M9 (1 ml) containing 10 µM cysteine (10 tubes) or 10 µM cysteine plus 1 mM Na2SO4 (10 tubes). The cell suspensions were incubated at 30 °C with reciprocal shaking for 1 h (five tubes) and 5 h (five tubes). Samples (100 µl) of each suspension were subjected to a
-galactosidase assay with o-nitrophenyl
-D-galactopyranoside as substrate (Miller, 1992
).
Analytical methods.
DMS, DMSO and DMSO2 accumulated in whole-cell reaction mixtures were detected with a gas chromatograph GC-14B (Shimadzu) fitted with a fused-silica chemically bonded capillary column DB-5 (0·258 mm inside diameter by 30 m, 1 µm film thickness; J&W Scientific). The head pressure of the helium carrier gas was 100 kPa. Each sample was injected into the column at 100 °C. After 2 min at 100 °C, the column temperature was increased to 200 °C at 10 °C min-1. The peaks of DMS, DMSO and DMSO2 were detected at 2·1 min, 4·8 min and 5·4 min, respectively.
Chemicals.
Chemicals were of the highest quality available and were purchased from Tokyo Kasei Kogyo or Kanto Chemical.
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RESULTS |
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Cloning and sequencing analysis of the Tn5-inserted region in Dfi74J
To identify the locus disrupted by Tn5 in Dfi74J, the region flanking the Tn5 insertion was cloned by transposon rescue techniques and sequenced. Sequencing of a 3·3 kb SphISacI fragment (Fig. 2) revealed that there were three ORFs (these were designated as sfnE, sfnC and sfnR, for dimethyl sulfone utilization), and that Tn5 was inserted at 166 bp upstream from the 3'-terminus of sfnR (Fig. 2
). The 2·9 kb region downstream from the SacI site was also cloned and sequenced. An ORF showing identity with glutathione S-transferase was located approximately 1·2 kb downstream of sfnR in the opposite direction (data not shown), but no ORFs were found immediately downstream of sfnR. All sfn genes were preceded by good consensus ribosome-binding sites (data not shown). The overall G+C content of the coding region was 65·2 mol%. The initial codons of sfnC and sfnR were located immediately downstream from the stop codons of the preceding ORFs (data not shown), indicating that the sfn gene cluster is probably transcribed as a transcriptional unit. By comparison with the
54-dependent consensus promoter (5'-TGGCACN5TTGCW-3') in E. coli (Reitzer & Schneider, 2001
), a signature of a
54-dependent promoter (5'-CGGCACGACGATTGCT-3'; underlined sequences are well conserved) was found 42 bp upstream from the initial codon of sfnE.
The deduced amino acid sequence of SfnE showed the highest identity (64 % amino acid identity) with MsuE from P. aeruginosa, which was reported to encode an NADH-dependent FMN reductase (Kertesz et al., 1999). SfnC is a 395 amino acid protein showing the highest identity (70 %) with MsuC (PA2355) from P. aeruginosa PAO1, whose function has not been fully elucidated (Kertesz et al., 1999
; Stover et al., 2000
; http://www.pseudomonas.com). However, SfnC also showed identity with two bacterial FMNH2-dependent monooxygenases: TdsC (47 %) from Paenibacillus sp. strain A11-2 (Ishii et al., 2000
) and DszC (43 %) from Rhodococcus sp. strain IGTS8 (Demone et al., 1994
), both of which catalyse stepwise monooxygenation of dibenzothiophene to dibenzothiophene sulfone.
SfnR exhibited identity with putative transcriptional regulators, PA2354 (78 %) and PA2359 (60 %), from P. aeruginosa PAO1 (Stover et al., 2000; http://www.pseudomonas.com). pa2354 is located immediately downstream of msuC (Stover et al., 2000
; http://www.pseudomonas.com), although it has not been confirmed that pa2354 is organized in an operon with the msuEDC operon of PAO1 (Kertesz et al., 1999
). Moreover, SfnR also showed identity with several bacterial NtrC-type response regulators such as NtrC (37 %) from Agrobacterium tumefaciens (accession no. I39719; Wardhan et al., 1989
), HydG (36 %) from Salmonella typhimurium (accession no. S19606; Chopra et al., 1991
), AtoC (36 %) from E. coli (accession no. B64992; Canellakis et al., 1993
), and TacA (34 %) from Caulobacter crescentus (accession no. AAC45640; Marques et al., 1997
). The sequence alignment of SfnR with these response regulators demonstrated that SfnR had the central ATP-dependent
54 factor (RpoN) interaction domain and the C-terminal helixturnhelix DNA-binding domain. However, SfnR was approximately 100 amino acids shorter than these response regulators, and did not have the N-terminal phospho-receiver domain, which is specific to the NtrC-type response regulators (data not shown). Therefore, SfnR appeared to participate in transcriptional regulation of unknown gene(s) by coordinating with
54-RNA polymerase.
Complementation of Dfi74J with sfnR
Dfi74J was complemented with a plasmid expressing sfnR to confirm that the DMSO2-utilization deficiency was caused by the disruption of sfnR. The 1966 bp HindIIISacI fragment containing the 3'-half of sfnC and the entire sfnR gene (Fig. 2) was cloned into pBBR1MCS-5 (Kovach et al., 1995
), to produce pBBRsfnR. Since the lac promoter is expressed constitutively in Pseudomonas species (Rist & Kertesz, 1998
), pBBRsfnR should express sfnR in Dfi74J. pBBRsfnR was introduced into Dfi74J by electrotransformation. pBBRsfnR restored the growth of Dfi74J on DMS, DMSO or DMSO2 (Table 2
), indicating that SfnR was essential for DMSO2 metabolism of strain DS1.
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pUCrpoN-Km containing the 873 bp SalISphI internal fragment of rpoN was constructed (see Methods). This plasmid was introduced into strain DS1 by electrotransformation, and a mutant
RPON (Kmr) was obtained (Fig. 3
a). Disruption of rpoN of
RPON was confirmed by PCR (Fig. 3b
). Mutant
RPON formed much smaller colonies on 2xYT plates than strain DS1 did, and the loss of motility was confirmed on swarm plates (data not shown). Also,
RPON could utilize glucose or ammonium chloride, but not succinate or nitrate, as carbon or nitrogen sources (data not shown). These phenotypic features of
RPON were in accordance with those of a P. putida rpoN mutant reported previously (Köhlter et al., 1989
). The
RPON mutant could utilize MSA, but not DMS, DMSO or DMSO2, as a sulfur source (Table 2
). The sulfur utilization selectivity of
RPON was the same as that of Dfi74J, suggesting that both SfnR and
54-RNA polymerase are involved in transcription of gene(s) for DMSO2 metabolism.
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Total RNAs were prepared from DS1 cells growing exponentially (0·3<OD550<0·7) in SFMM containing DMSO, Na2SO4, or DMSO plus Na2SO4. The total RNAs (10 µg) were subjected to Northern hybridization with probes specific to sfnC or sfnR (Fig. 2). An approximately 3·0 kb band hybridizing with an sfnC or sfnR probe was detected only for total RNA from DMSO-grown cells; the size corresponded to the length of the sfnECR-coding region (Fig. 4
a). To confirm the co-transcription of sfnR and sfnC genes, RT-PCR was done using total RNAs from sulfate- or MSA-grown DS1 cells and a set of primers, rt-sfnC-FW and rt-sfnR-RV, designed on the 3'-terminus of sfnC and 5'-terminus of sfnR, respectively (Table 1; Fig. 2
). A 400 bp amplified product was observed only in the reaction mixture containing total RNA from MSA-grown cells (Fig. 4b
). These findings indicated that the sfnECR gene cluster was organized as an SSI operon, and that conversion of DMSO2 to MSA occurs only under sulfate limitation in strain DS1.
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Expression of the sfnECR operon requires the LysR-type regulator, CysB
It has been suggested that a P. aeruginosa cysB mutant does not utilize MSA probably because of the loss of expression of the msuEDC and ssuEADCBF operons (Kertesz et al., 1999). Also, since the sfnECR operon was induced under sulfate limitation, expression of the sfn operon was suspected to be under the control of CysB.
To examine the involvement of CysB in the expression of the sfn operon, a cysB mutant of strain DS1, CYSB, was constructed by integration of pBS
cysB-Km (see Methods) into the chromosomal cysB by single-crossover recombination (data not shown). The sfn promoter activity of strain DS1(pMEsfn-lacZ) was then compared with that of
CYSB(pMEsfn-lacZ).
CYSB could grow on cysteine, but not on sulfate, as a sole sulfur source (data not shown), which was in accordance with the characteristics of the P. aeruginosa cysB mutant reported previously (Kertesz et al., 1999
). The
-galactosidase activities of
CYSB(pMEsfn-lacZ) were 10-fold and 4·1-fold lower than those of strain DS1(pMEsfn-lacZ) after 1 h and 5 h under the sulfate-starvation condition, while those of DS1(pMEsfn-lacZ) and
CYSB(pMEsfn-lacZ) were at similar levels in the presence of sulfate (Fig. 5
). This suggested that CysB played an important role in the SSI expression of the sfnECR operon.
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DISCUSSION |
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The sfnECR operon of strain DS1 showed identity with the msuEDCpa2354 gene cluster of P. aeruginosa PAO1 at the DNA level (more than 80 % similarity). pa2354 is also predicted to be part of an operon with msuEDC and to participate in DMSO2 metabolism of PAO1, because of its ability to grow on DMSO as a sulfur source (Hummerjohann et al., 1998). Also, it is suggested that operons sfnECR and msuEDCpa2354 come from a common ancestor. However, interestingly, a gene corresponding to msuD, whose product catalyses desulfonation of MSA in P. aeruginosa (Kertesz et al., 1999
), was lacking in the sfnECR operon of strain DS1 (Fig. 2
). Kertesz et al. (1999)
have reported that disruption of msuD does not affect the capacity of P. aeruginosa strain PAO1 to grow on MSA, probably due to the presence of ssuD, whose product shows 72 % amino acid identity with MsuD (Kahnert et al., 2000
). By contrast, disruption of ssuD in P. putida strains S-313 and DS1 led to loss of the capacity to grow on MSA (Kahnert et al., 2000
; Endoh et al., 2003
), indicating that ssuEADCBF encodes the sole MSA desulfonation system on P. putida genome. Hence, it is speculated that sulfonate desulfonation systems in P. putida were rearranged in the evolutionary process.
Transcriptional regulators involved in organosulfur assimilation in E. coli or Pseudomonas spp.
So far, several transcriptional regulators involved in expression of SSI organosulfur-assimilating genes have been identified and investigated in E. coli and Pseudomonas spp. In E. coli, two LysR-type transcriptional regulators (CysB and Cbl) control the expression of tauABCD and ssuEADCB under sulfate limitation; these operons are required for taurine and alkanesulfonate utilization, respectively (van der Ploeg et al., 1996, 1999
; Bykowski et al., 2002
). Although cysB plays an essential role in organosulfur assimilation in P. aeruginosa (Kertesz et al., 1999
), a gene corresponding to the E. coli cbl was not found in the P. aeruginosa genome (Stover et al., 2000
; http://www.pseudomonas.com). This implies that the SSI genes of Pseudomonas sp. are regulated differently from those of E. coli. Alternatively, three genes, sdsB, asfR and sftR, encoding LysR-type transcriptional regulators have been identified in Pseudomonas sp. (Davison et al., 1992
; Vermeij et al., 1999
; Kahnert et al., 2002
). SdsB has been reported to activate expression of sdsA, whose product catalyses desulfation of SDS in Pseudomonas sp. ATCC 19151 (Davison et al., 1992
). AsfR modulates transcription of the asfABC operon, whose products (AsfAB but not AsfC) are required for arylsulfonate utilization of P. putida S-313 (Vermeij et al., 1999
). SftR activates expression of a gene cluster, atsBC, atsRK and sftPastA, whose products play an essential role in utilization of aryl- or alkylsulfate esters in P. putida S-313 (Kahnert et al., 2002
). Accordingly, SfnR identified in strain DS1 is the fourth transcriptional regulator involved in organosulfur assimilation in Pseudomonas sp. However, unlike these LysR-type regulators, SfnR is related to NtrC-type transcriptional regulators, suggesting that the mechanism of transcriptional activation is quite different from that of LysR-type regulators. LysR-type regulators usually activate the expression of target genes by interacting with
70-RNA polymerase, whereas NtrC-type regulators employ
54-RNA polymerase (Rombel et al., 1998
; Ramos et al., 1997
). To our knowledge this is the first report that a
54-dependent transcriptional regulator is involved in sulfur assimilation.
Relationship between 54 and sulfur assimilation
The 54 factor has been generally considered as a sigma factor for nitrogen metabolism in E. coli, whereas P. putida
54-RNA polymerase controls a number of disparate functions including nitrogen metabolism, growth on some carbon sources (Köhlter et al., 1989
), polar flagellar synthesis (Pandza et al., 2000
), and degradation of hydrocarbons such as m-xylene (Ramos et al., 1997
) and phenols (Shingler et al., 1993
). However, the relationship between the
54 factor and sulfur metabolism in Gram-negative bacteria has not been reported, probably due to its dispensability for sulfur amino acid biosynthesis. Cases & Lorenzo (2001)
proposed that the
54 factor of P. putida plays important roles in surviving environmental stress. Thus, the involvement of P. putida
54 factor in SSR is not surprising, because sulfate-limited stress is thought to be ubiquitous in aerobic forest and agricultural soils inhabited by pseudomonads due to the abundance of organosulfur compounds such as sulfonates and sulfate esters rather than inorganic sulfate (Fitzgerald, 1976
; Autry & Fitzgerald, 1990
).
Transcriptional regulation of the sfnECR operon in P. putida strain DS1
CysB has been suggested to be involved in transcription of organosulfur-assimilating genes in P. aeruginosa (Kertesz et al., 1999; Hummerjohann et al., 2000
). However, direct evidence that CysB activates expression of SSI genes has not been obtained, because the P. aeruginosa cysB mutant can utilize only preferred sulfur compounds such as cysteine and thiosulfate, which do not induce SSR in pseudomonads. We here obtained direct evidence that CysB protein is involved in activation of transcription of the sfnECR operon by a reporter gene assay with supplementation of a small amount of cysteine, which does not prevent the SSR (Fig. 5
). However, interestingly, a signature of a
54-dependent promoter was found upstream of the sfnE site. Since sfnR deficiency did not affect its own expression under sulfate limitation (Fig. 4b
), it was suggested that sfnECR is under the control of an unknown
54-dependent regulator. To elucidate that, detailed examination of the transcriptional mechanism of sfn promoter will be needed.
The regulatory model of DMSO2 assimilation in P. putida
The cascade regulatory model of DMSO2 metabolism of strain DS1 is proposed as follows. Under sulfate limitation, CysB functions as a master regulator for organosulfur-assimilating genes, and activates expression of an unknown 54-dependent regulator. The unknown
54-dependent regulator enhances the expression of the sfnECR operon. Then, the resultant SfnR activates the expression of gene(s) involved in DMSO2 metabolism and interacting with
54-RNA polymerase. In this process, unlike NtrC-type transcriptional regulators, SfnR probably functions only as a transcriptional activator rather than a response regulator, because of the lack of the N-terminal phospho-receiver domain. On the other hand, since Dfi74J grows normally on taurine, alkylsulfonates and sulfate esters (Endoh et al., 2003
), SfnR appears not to be involved in transcriptional regulation of the ssu, tau and ats SSI operons. To obtain direct evidence for the above hypothesis, we are attempting to clone the gene(s) involved in DMSO2 conversion.
Is an SfnEC two-component monooxygenase system involved in DMS catabolism?
It is possible that an SfnEC two-component monooxygenase system might be involved in DMS catabolism. sfnEC was transcribed together with sfnR, and SfnC showed identity (43 %) with DszC from Rhodococcus sp. IGTS8, catalysing stepwise monooxygenation of the sulfur atom of dibenzothiophene to dibenzothiophene sulfone (Denome et al., 1994), which is quite similar to oxidation of DMS to DMSO2 in strain DS1 (Endoh et al., 2003
). To elucidate our hypothesis, we are also attempting to characterize the SfnEC monooxygenase system.
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ACKNOWLEDGEMENTS |
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Received 1 October 2002;
revised 7 January 2003;
accepted 9 January 2003.
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