Abstract
We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a Mr value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.
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References
Akamatsu T, Sekiguchi J (1987) Genetic mapping by means of protoplast fusion in Bacillus subtilis. Mol Gen Genet 208:254–262
Archibald AR (1989) The Bacillus cell envelope. In: Harwood CR (ed) Bacillus. Plenum Press, New York, pp 217–254
Croux C, Garcia JL (1991) Sequence of the lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes. Gene104:25–31
Duez C, Lakaye B, Houba S, Dusart J, Ghuysen J-M (1990) Cloning, nucleotide sequence and amplified expression of the gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G. FEMS Microbiol Lett 71:215–220
Fein JE, Rogers HJ (1976) Autolytic enzyme deficient mutants of Bacillus subtilis 168. J Bacteriol 127:1427–1442
Foster SJ (1991) Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2. J Gen Microbiol 137:1987–1998
Ghuysen JM, Tipper DJ, Strominger JL (1966) Enzymes that degrade bacterial cell walls. Methods Enzymol 8:685–699
Hourdou M-L, Duez C, Joris B, Vacheron M-J, Guinand M, Michel G, Ghuysen J-M (1992) Cloning and nucleotide sequence of the gene encoding the γ-d-glutamyl-l-diamino acid endopeptidase II of Bacillus sphaericus. FEMS Microbiol Lett 91:165–170
Joris B, Beeumen JV, Casagrande F, Gerday C, Frère J-M, Ghuysen J-M (1983) The complete amino acid sequence of the Zn2+-containing d-alanyl-d-alanine-cleaving carboxypeptidase of Streptomyces albus G. Eur J Biochem130:53–69
Kuroda A, Sekiguchi J (1990) Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. J Gen Microbiol 136:2209–2216
Kuroda A, Sekiguchi J (1991) Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. J Bacteriol 173:7304–7312
Kuroda A, Imazeki M, Sekiguchi J (1991) Purification and characterization of a cell wall hydrolase encoded by the cwlA gene of Bacillus subtilis. FEMS Microbiol Lett 81:9–14
Kuroda A, Sugimoto Y, Funahashi T, Sekiguchi J (1992) Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase. Mol Gen Genet 234:129–137
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lazarevic V, Margot P, Soldo B, Karamata D (1992) Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-l-alanine amidase and its modifier. J Gen Microbiol 138:1949–1961
Lee JK, Edwards CW, Hulett FM (1991) Identification of four unique clones encoding 10 kDa proteins from Bacillus that cause phenotypic complementation of a phoA mutant strain of Escherichia coli. J Gen Microbiol 137:667–677
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Matsuda T, Kotani S, Kato K (1968) Structure of the cell walls of Lactobacillus plantarum, ATCC8014. 1. Isolation and identification of the peptides released from cell wall peptidoglycans by Streptomyces L-3 enzyme. Biken J 11:111–126
Potvin C, Leclerc D, Tremblay G, Asselin A, Bellemare G (1988) Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Mol Gen Genet 214:241–248
Robson RL, Baddiley J (1977) Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant. J Bacteriol 129:1051–1058
Rogers HJ, Perkins HR, Ward JB (1980) Microbial cell walls and membranes. Chapman and Hall, London
Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, 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
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Oda, Y., Nakayama, R., Kuroda, A. et al. Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis . Molec. Gen. Genet. 241, 380–388 (1993). https://doi.org/10.1007/BF00284691
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DOI: https://doi.org/10.1007/BF00284691