The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate

Abstract

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

Functional Dissection of the LysR-type CysB Transcriptional Regulator

“…1) that may contribute to inducer binding and response. Non-inducible CysB variants were unable to respond to the inducer, acetylserine, by the conformational change that allows the transition of the slow DNA-protein complex to the fast complex, of which only the latter represents the structure required for transcriptional activation of the cysP promoter (16,18). The interaction of these CysB variants with the cysB promoter region was also unaffected by acetylserine, in contrast to wild-type CysB, whose binding to p cysB was inhibited by the inducer.…”

“…The sizes of two major runoff products, 73 and 66 nucleotides, corresponded to transcription start sites at positions Ϫ1 and ϩ6, respectively, relative to the major in vivo start site of the E. coli cysP promoter. The appearance of multiple transcripts in the runoff experiment using the E. coli cysP promoter region is not surprising, as it was also observed with the S. typhimurium cysP promoter, in which two major runoff products (corresponding to transcription start sites at positions Ϫ2 and ϩ5 relative to the in vivo transcription start site) and several minor products were synthesized (18). In contrast to WT CysB, the CysB Y27G mutant protein showed very low activity in an analogous runoff assay (Fig.…”

“…Examples of LysR family members, NahR (7), OxyR (9), and GcvA (36), also suggest the correlation between the oligomerization defect and the inability of the mutant protein to exert a dominant-negative effect (also called a "poisoning effect") on the activity of the wild-type counterpart produced by the cell. For CysB, the negative dominance was clearly seen with some non-repressing (non-binding) variants (with mutations in region [11][12][13][14][15][16][17][18][19][20][21][22] as well as with non-inducible variants (Fig. 1).…”

“…The complexes formed by E. coli WT CysB with p cysP , shown in Fig. 4A, could be identified by strict correspondence to those formed by highly purified S. typhimurium CysB, analyzed in detail previously (16,18). When a single CysB tetramer binds to p cysP , it induces DNA bending of ϳ100°in the absence of acetylserine and of ϳ50°in its presence, giving rise to "slow" (C1s) and "fast" (C1f) primary complexes, respectively.…”

“…In this case, acetylserine reduces binding of CysB to DNA, thereby releasing the repression of the cysB gene. Sulfur limitation required for high-level expression of cys genes in vivo can be explained by the fact that sulfide and thiosulfate act as anti-inducers, competing with N-acetylserine for the CysB activator (18,19). In addition, cysteine is an inhibitor of serine transacetylase, the enzyme required for acetylserine biosynthesis.…”