Abstract
Mechanisms of gene regulation have not yet been extensively studied in thermophilic bacteria. In previous studies we showed that theBacillus stearothermophilus argCJBD gene cluster is subject to specific repression by arginine. Here we report the cloning by colony hybridization, and characterization of the proximal part of theargC gene together with the adjacent control region of the cluster. The promoter was identified by primer extension mapping of theargC transcription startpoint: a sequence overlapping it was found to be similar to the arginine operators ofB. subtilis and to a smaller extent ofE. coli. Use of anargC-lacZ gene fusion revealed that theargC promoter is strongly repressed by the heterologousB. subtilis arginine repressor/activator AhrC inE. coli cells. Mobility shift and DNase I footprinting experiments revealed tight, specific and arginine-dependent binding of this operator-like sequence to purified AhrC. It is there-fore very likely that inB. stearothermophilus the expression of theargCJBD operon is modulated by a repressor that is the thermophilic homologue of AhrC.
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References
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1993) Current protocols in molecular biology. John Wiley and Sons, New York
Blakely G, May G, McCulloch R, Arciszewska LK, Burke M, Lovett ST, Sherratt DJ (1993) Two related recombinases are required for site-specific recombination atdif andcer inEscherichia coli K12. Cell 75:351–361
Charlier D, Roovers M, Van Vliet F, Boyen A, Cunin R, Nakamura Y, Glansdorff N, Piérard A (1992) Arginine regulon ofEscherichia coli K-12. A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression. J Mol Biol 226:367–386
Cunin R, Glansdorff N, Piérard A, Stalon V (1986) Biosynthesis and metabolism of arginine in bacteria. Microbiol Revs 50:314–352
Czaplewski LG, North AK, Smith MCM, Baumberg S, Stockley PG (1992) Purification and initial characterization of AhrC the regulator of arginine metabolism genes inBacillus subtilis. Mol Microbiol 6:267–275
Galas DJ, Schmitz A (1978) DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5:3157–3170
Glansdorff N (1996) Biosynthesis of arginine and polyamines. In: Neidhardt FC (ed in chief)Escherichia coli andSalmonella: cellular and molecular biology. American Society for Microbiology Press, Washington, DC, pp 408–433
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–199
Klingel U, Miller CM, North AK, Stockley PG, Baumberg S (1995) A binding site for activation by theBacillus subtilis AhrC protein, a repressor/activator of arginine metabolism. Mol Gen Genet 248:329–340
Legrain C, Demarez M, Glansdorff N, Piérard A (1995) Ammonia-dependent synthesis and metabolic channeling of carbamoyl phosphate in the hyperthermophilic archaeonPyrococcus furiosus. Microbiology 141:1093–1099
Lim D, Oppenheim JD, Eckhardt T, Maas WK (1987) Nucleotide sequence of theargR gene ofEscherichia coli K12 and isolation of its product, the arginine repressor. Proc Natl Acad Sci USA 84:6697–6701
Lu CD, Houghton JE, Abdelal AT (1992) Characterization of the arginine repressor fromSalmonella typhimurium and its interactions with thecarAB operator. J Mol Biol 225:11–24
Maas WK (1994) The arginine repressor ofEscherichia coli. Microbiol Rev 58:631–640
Miller JH (1992) A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Mountain A, Mann NM, Munton RN, Baumberg S (1984) Cloning of aBacillus subtilis restriction fragment complementing auxotrophic mutants of eightEscherichia coli genes of arginine biosynthesis. Mol Gen Genet 197:82–89
Mountain A, McChesney J, Smith MCM, Baumberg S (1986) Gene sequence encoding early enzymes of arginine synthesis within a cluster inBacillus subtilis, as revealed by cloning inEscherichia coli. J Bacteriol 165:1026–1028
North AK, Smith MCM, Baumberg S (1989) Nucleotide sequence of aBacillus subtilis arginine regulatory gene and homology of its product to theEscherichia coli arginine repressor. Gene 80:29–38
Saito H, Miura, K-I (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Sakanyan VA, Hovsepyan AS, Mett IL, Kochikyan AV, Petrosyan PK (1990) Molecular cloning and structural-functional analysis of the arginine biosynthesis genes of the thermophilic bacteriumBacillus stearothermophilus. Genetika (USSR) 26:1915–1925
Sakanyan V, Kochikyan A, Mett I, Legrain C, Charlier D, Piérard A, Glansdorff N (1992) A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilicBacillus species. J Gen Microbiol 138:125–130
Sakanyan V, Charlier D, Legrain C, Kochikyan A, Mett I, Piérard A, Glansdorff N (1993a) Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis inBacillus stearothermophilus: dual function of ornithine acetyltransferase. J Gen Microbiol 139:393–402
Sakanyan V, Legrain C, Charlier D, Kochikyan AV, Osina NK, and Glansdorff N (1993b)N-acetylglutamate-5-phosphotransferase of thermophilic bacteriumBacillus stearothermophilus: nucleotide sequence and enzyme characterization. Genetika (Russia) 29:556–570
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Smith MCM, Czaplewski L, North AK, Baumberg S, Stockely PG (1989) Sequences required for regulation of arginine biosynthesis promoters are conserved betweenBacillus subtilis andEscherichia coli. Mol Microbiol 3:23–28
Smith MCM, Mountain A, Baumberg S (1990) Nucleotide sequence of theBacillus subtilis argC gene encodingN-acetylglutamate-γ-semialdehyde dehydrogenase. Nucleic Acids Res 18:4595
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stirling CJ, Szatmari G, Stewart G, Smith MCM, Sherratt DJ (1988) The arginine repressor is essential for plasmid-stabilising site-specific recombination of the ColE1cer locus. EMBO J 7:4389–4395
Tian G, Lim D, Carey J, Maas WK (1992) Binding of the arginine repressor ofEscherichia coli K12 to its operator sites. J Mol Biol 226:387–397
Van de Casteele M, Demarez M, Legrain C, Glansdorff N, Piérard A (1990) Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria. J Gen Microbiol 136:1177–1183
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Communicated by A. Kondorosi
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Savchenko, A., Charlier, D., Dion, M. et al. The arginine operon ofBacillus stearothermophilus: characterization of the control region and its interaction with the heterologousB. subtilis arginine repressor. Molec. Gen. Genet. 252, 69–78 (1996). https://doi.org/10.1007/BF02173206
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DOI: https://doi.org/10.1007/BF02173206