The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH 2 -dependent monooxygenase and an NAD(P)Hdependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639 -26646). Synthesis of -galactosidase from a transcriptional chromosomal ssuE-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysRtype transcriptional regulator. Electrophoretic mobility shift assays with the ssu promoter region and measurements of -galactosidase from plasmid-encoded ssuElacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the ؊35 region. CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE-lacZ fusion although the ssu promoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE-lacZ fusion.
The nucleotide sequence of the sulfate and thiosulfate transport gene cluster has been determined and located 3' to the gene (cysP) encoding the thiosulfate-binding protein. Four open reading frames, designated cysT, cysW, cysA, and cysM, have been identified. Similarities in primary structure were observed between (i) the deduced amino acid sequences of CysT and CysW with membrane-bound components of other binding protein-dependent transport systems, (ii) that of the CysA sequence with the "conserved" component of such systems, and (iii) that of the CysM sequence with O-acetylserine sulfhydrylase A (cysK gene product) and the Is-subunit of tryptophan synthase (coded by trpB). Expression of the four genes was analyzed in the T7 promoter-polymerase system.
The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine. N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Assimilatory sulfate reduction in Salmonella typhimurium and Escherichia coli commences with the uptake of extracellular sulfate, a process requiring a periplasmic transport system termed the sulfate permease system (7,8,43). All but one of the components of the sulfate permease system are encoded by contiguous genes located at 52 min on the E. coli map (3, 20) and at 49 min on the S. typhimurium map (8, 48), which in S. typhimurium were originally designated cysAa, cysAb, and cysAc (31). This genetic region has recently been cloned and sequenced in E. coli and found to contain five open reading frames, which, beginning furthest upstream, were designated cysP, cysT, cysW, cysA, and cysM (13, 50). cysM encodes O-acetylserine (thiol)-lyase B, which catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide (4, 15) and also the synthesis of S-sulfocysteine from O-acetyl-L-serine and thiosulfate (35,36). The deduced amino acid sequences of E. coli cysT, cysW, and cysA suggest they encode the three membrane-bound components that are typical of periplasmic substrate-binding transport systems (2), and these three genes probably correspond to S. typhimurium cysAa, cysAb, and cysAc, respectively. cysP has been shown to encode a periplasmic thiosulfate binding protein (13) that is similar to but distinct from the sulfate binding protein from S. typhimurium (9,16,42,43). Since
The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.Genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli are positively regulated by the CysB protein (13, 15, 16), a tetramer of identical 36-kDa subunits (20,24) that is a member of the LysR family of bacterial activator proteins (9). In addition to requiring CysB protein, derepression of this biosynthetic pathway requires sulfur limitation and the L-cysteine precursor O-acetyl-L-serine, which acts as an inducer (14,15). N-Acetyl-L-serine is also an inducer (25) and is derived nonenzymatically from O-acetyl-L-serine by an intramolecular O-to-N-acetyl shift (6). The requirement for sulfur limitation appears to be due to an anti-inducer effect of sulfide, which interferes with the ability of CysB protein and inducer to activate gene expression both in vivo and in vitro (14,15,26).In vitro studies have shown that transcription initiation from the cysJIH promoter requires CysB and either N-acetyl-L-serine or O-acetyl-L-serine (25) promoter activity. Evidence is also presented that indicates that binding of CysB protein to the cysK promoter induces DNA bending in the absence of inducer but little or no bending in the presence of inducer. MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli JM105 was the transformation recipient for isolating pUC19 derivatives (36) and the host for M13 phage propagation. NM522 [hsdA5 A(lac-pro)(F' pro' lacIqZAMJ5)] was the host for pT7T3 phagemid derivatives. S. typhimurium strains included the wild-type LT2 st...
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