Article

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli

Journal of Biological Chemistry (Impact Factor: 4.57). 06/1987; 262(13):5999-6005.
Source: PubMed

ABSTRACT

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.

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    • "Interaction of CmbR with P metC in L. lactis are dimers (Schell, 1993). The CysB proteins of E. coli and S. enterica serovar Typhimurium are tetramers of identical 36 kDa subunits (Ostrowski et al., 1987). By analogy, it seems likely that CmbR acts as a multimer, possibly as a tetramer. "
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    ABSTRACT: The metC-cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the metC promoter. This binding was stimulated by O-acetyl-L-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter.
    Full-text · Article · Mar 2005 · Microbiology
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    • "Conditions for probe preparation, DNA-binding reactions and D(argF-lac)U169 D(his-gnd) thi-1 Thomas and Glass (1991) rpsL150 zhc::Tn10 WAM105 F – araD139 D(argF-lac)U169 D(his-gnd) thi-1 Thomas and Glass (1991) rpsL150 gltSo flbB301 relA1 deoC rbsR rpoA341 GS1129 MC4100 pheA905 l(sulA::lacZ) G . Stauffer Plasmids pGEM® T Easy Vector for cloning PCR fragments, Ap R Promega pJOH1 cysB E. coli cloned in pBR322, Ap R Ostrowski et al. (1987) pMH199 a Promoterless cysB cloned in pTrc99A, expressing Lochowska et al. (2001) CysB wt from trc promoter pMH235 Derivative of pMH199 encoding CysB Y27G Lochowska et al. (2001) pMH247 b cysB cloned in pTrc99A expressing CysB wt –His 6 This study from trc promoter pMH1822 E. coli cysPT¢ region cloned on EcoRV fragment Laboratory collection in pUC18 HincII, Ap R pLAW2 Plasmid carrying rpoA encoding RNAP a subunit Zou et al. (1992) pLAW2phs pLAW2 with mutation rpoA341 (K271E in aCTD) M. Thomas pHTf1 Plasmids encoding RNAP a subunits with Tang et al. (1994); derivatives alanine substitutions at positions 255–271 Gaal et al. (1996) pREII a Plasmids encoding RNAP a subunits with Blatter et al. (1994);Gaal et al. derivatives alanine substitutions at positions 273–329 (1996); Kainz and Gourse (1998) pSR Cloning vector, l oop terminator, pBR322 origin Kolb et al. (1995) pTrc99A Expression vector, Ap r , trc promoter, pBR322 origin Pharmacia pTB23 cysP promoter on EcoRI–HindIII fragment in pSR This study pDL804 c Derivative of pACYC177 encoding Dmitrova et al. (1998) LexA 1-87 408–Jun zipper fusion protein pMS604 d Derivative of pBR322 encoding Dmitrova et al. (1998) LexA 1-87 WT–Fos zipper fusion protein Plasmids constructed in this study: a. Derivatives of pMH199 encoding CysB with single residue substitutions are listed inTable 1 DNase I digestions were as described previously (Hryniewicz and Kredich, 1991). The E. coli cysP promoter fragment (from position -321 to +72 relative to tsp) was amplified by PCR with primers ECCP1 and ECCP2, the latter being 5¢ endlabelled with [g-32 P]-ATP using polynucleotide kinase. "
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    ABSTRACT: CysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the alpha2 and alpha3 helices forming an N-terminal helix-turn-helix motif. On the basis of the characteristics of alanine-substituted mutants, we propose that CysB residues Y27, T28 and S29, lying in this turn region, comprise an 'activating region' (AR) that is crucial for positive control of the cysP promoter, but not for DNA binding and inducer response activities of CysB. Using a library of alanine substitutions in the C-terminal domain of the RNAP alpha subunit (alpha-CTD), we identify several residues in alpha-CTD that are important for CysB-dependent transcription from the cysP promoter. After probing potential protein-protein contacts in vivo with a LexA-based two-hybrid system, we propose that the '273 determinant' on alpha-CTD, including residues K271 and E273, represents a target for interaction with CysB at the cysP promoter.
    Full-text · Article · Sep 2004 · Molecular Microbiology
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    • "Plasmids pRS415 lacZYA + promoterless vector used for construction of lacZ operon fusions , Amp R Simons et al . ( 1987 ) pMS421 Low - copy - number cloning vector , Spc R Grana et al . ( 1988 ) pQE70 Expression vector , Amp R Qiagen pREP4 Low - copy plasmid , carries lacI gene , Kan R Qiagen pJOH1 pBR322 carrying WT cysB , Amp R Ostrowski et al . ( 1987 ) pHV3002 pMS421 carrying WT hslJ on 3 ? 7 kb DNA fragment from l265 Lilic et al . ( 2003 )"
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    ABSTRACT: The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium. cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance. In this study the hslJ transcription start point and the corresponding putative sigma(70) promoter were determined. The hslJ promoter region was defined by employing different hslJ-lacZ operon fusions, and transcription of the hslJ gene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor of hslJ transcription and binds the hslJ promoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligand N-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression of hslJ transcription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.
    Full-text · Article · Jan 2004 · Microbiology
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