Sulfate and thiosulfate transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved in thiosulfate binding

Abstract

The sequence of 1,973 nucleotides encompassing the region at and directly adjacent to the CysB-dependent promoter controlling expression and synthesis of the sulfate-thiosulfate transport system of Escherichia coli has been determined. The transcription start site has been mapped by primer Escherichia coli and Salmonella typhimurium possess a sulfate transport system which is controlled in parallel with cysteine-biosynthetic enzymes and which is a part of the cysteine regulon (19); gene expression of this region requires sulfur limitation and the positive regulator CysB, the product of the cysB gene. Mutations impairing sulfate transport are localized in the "cysA" region, which maps at min 49 of S. typhimurium (8, 9, 25) and min 52 of the E. coli chromosome (4, 18). In E. coli a second locus, cysZ, situated about 10 kilobases (kb) from "cysA," has been reported to be essential for sulfate transport (34). Early genetic studies on the "cysA" locus in S. typhimurium showed the presence of three complementation groups (25). Furthermore, these studies indicated that integrity of the whole three-cistron region is required for both sulfate and thiosulfate transport (8). Subsequently, the specific sulfate-binding protein (SBP) was isolated from S. typhimurium (32) and studied in detail (16,29,33,35). This analysis raised the possibility that the sulfate transport system might have similarities with other well-characterized binding-protein-dependent transport systems (1), where a single operon contains genes encoding the binding protein and several membrane-bound components. However, a direct relationship between SBP and the genetics of sulfate transport remains to be demonstrated because (i) none of the three "cysA" cistrons of S. typhimurium has been shown to encode this activity, and (ii) transportdefective mutants with a specific lesion in SBP could not be isolated despite a thorough search (29).We have cloned the region containing the "cysA" and cysM (the latter specifying the O-acetylserine sulfhydrylase B) loci of E. coli (40) Acta Biochim. Polon., in press). The sequence analysis of this region presented in the accompanying paper (41) revealed the presence of four open reading frames (ORFs) coding for three membrane components of the sulfate transport system and of an ORF for cysM. Here, we report the identification of an unknown locus, designated cysP, specifying the first gene of the region encoding the sulfatethiosulfate transport system. It is expressed from a CysBdependent promoter and encodes a thiosulfate-binding protein.(The nucleotide sequence data reported here will appear in the EMBL, GenBank, and DDBJ nucleotide sequence data bases under accession no. M32101.) MATERIALS AND METHODSBacterial strains and phages. All strains used in this study were derivatives ofE. coli K-12 and S. typhimurium LT2 and are listed in

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

“…The DNA sequence of the S. typhimurium cysP coding region and the 117 bp preceding the ATG start codon has been reported previously (13). An additional upstream sequence was obtained from pMHK1, which is a pT7T318U derivative containing the cysP promoter and part of the cysP coding region from S. typhimurium on a 1.07-kb BamHIEcoRI fragment.…”

“…An apparent rho-independent terminator located between cysA and cysM indicates that the later may be expressed from its own promoter. An in vivo transcription start site for the E. coli cysP promoter has been demonstrated with primer extension analysis (13). In this report we identify the in vivo start site for the S. typhimurium cysP promoter and characterize in vitro interactions of this promoter with the CysB protein, O-acetyl-L-serine, and N-acetyl-L-serine.…”

“…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 (34). DNA bending at the cysK promoter requires the presence of two adjacent CysB protein binding sites and has not been observed with the cysJIH promoter, which has only a single binding site.…”

“…The presence in E. coli of overlapping start and termination codons between cysP, cysT, and cysW, a small overlap of the coding regions of cysW and cysA (50), and the polar effect of a cysP mutation (13) suggest that these four genes comprise a single operon, which is expressed from the cysP promoter. An apparent rho-independent terminator located between cysA and cysM indicates that the later may be expressed from its own promoter.…”

“…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).…”