Cloning and characterization of the gene encoding penicillin-binding protein A of Streptomyces griseus

• Published in: FEMS microbiology letters 2000-12, Vol.193 (1), p.63-68
• Main Authors: Jiang, Hao, Kendrick, Kathleen E.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Amino acids; Antibiotics; Apramycin; Bacterial Proteins; Bacteriology; Base Sequence; Biological and medical sciences; Blotting, Southern; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - physiology; Chromosomes; Cloning; Cloning, Molecular; Deoxyribonucleic acid; DNA; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetics; Hexosyltransferases; Homology; Hybridization; Microbiology; Molecular Sequence Data; Muramoylpentapeptide Carboxypeptidase - chemistry; Muramoylpentapeptide Carboxypeptidase - genetics; Muramoylpentapeptide Carboxypeptidase - physiology; Mutagenesis, Insertional; PbpA gene; Penicillin; Penicillin-binding protein; Penicillin-binding protein A; Penicillin-Binding Proteins; Peptidyl Transferases; Phenotype; Polymerase Chain Reaction; Protein A; Proteins; Sonication; Spores, Bacterial - physiology; Sporulation; Streptomyces griseus; Streptomyces griseus - chemistry; Streptomyces griseus - genetics; Streptomyces griseus - physiology
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2000.tb09403.x

Abstract An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.