Dataset Information

Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene.

ABSTRACT: A major Bacillus subtilis 168S autolysin (N-acetylmuramoyl-L-alanine amidase [EC 3.5.1.28]) was purified and then cleaved with cyanogen bromide. The N-terminal amino acid sequence of one of the resultant peptides was determined in order to make synthetic oligonucleotides. A 2.5-kb EcoRI fragment was cloned into Escherichia coli JM109 and detected by colony hybridization by using the oligonucleotides as probes. Sequencing of the insert showed the presence of an open reading frame (designated cwlB), starting at a UUG codon, which encodes a polypeptide of 496 amino acids with a molecular mass of 52,623 Da. CWLB had a presumed signal peptide which is processed after Ala at position 24. Insertional inactivation of the cwlB gene of the B. subtilis chromosome led to an approximately 90% decrease in the total cell wall hydrolytic activity of stationary-phase cells and extraordinary resistance to cell lysis, even after 6 days of incubation at 37 degrees C. No apparent changes in cell morphology, motility, competence, sporulation, or germination were observed.

SUBMITTER: Kuroda A

PROVIDER: S-EPMC209238 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene.

Kuroda A A Sekiguchi J J

Journal of bacteriology 19911101 22

A major Bacillus subtilis 168S autolysin (N-acetylmuramoyl-L-alanine amidase [EC 3.5.1.28]) was purified and then cleaved with cyanogen bromide. The N-terminal amino acid sequence of one of the resultant peptides was determined in order to make synthetic oligonucleotides. A 2.5-kb EcoRI fragment was cloned into Escherichia coli JM109 and detected by colony hybridization by using the oligonucleotides as probes. Sequencing of the insert showed the presence of an open reading frame (designated cwlB ...[more]

PMID: 1682302

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Project description:The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456-7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitive Escherichia coli psd-2 mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the psd-2 mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of 14C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in E. coli psd-2 cells harboring a Bacillus psd plasmid. E. coli cells harboring the psd plasmid, the expression of which was under the control of the T7phi10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together with pss, was located by Southern hybridization to the 238- to 306-kb SfiI-NotI fragment of the chromosome. A B. subtilis strain harboring an interrupted psd allele, psd1::neo, was constructed. The null psd mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for the E. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss and psd mutants, glucosyldiacylglycerol content increased two- to fourfold. The results suggest that the lack of phosphatidylethanolamine in the B. subtilis membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism.

Dataset Information

Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene.

Publications

Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene.

Similar Datasets

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