Molecular analysis of penicillin-binding proteins and a gene cluster in Streptomyces griseus.

摘要

Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85 kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Treatment with cefoxitin, a β-lactam antibiotic, prevented septum formation during sporulation but not during vegetative growth. Fluorescein-tagged β-lactam antibiotics were used to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. The 85 kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation.; An internal segment of the penicillin-binding protein gene, pbpA, of S. 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. No obvious defects in growth, sporulation, or spore sonication resistance were observed in pbpA null mutants, suggesting that PBP A is not essential for growth and sporulation under normal laboratory conditions.; A cefoxitin-plus-sonication-resistant mutant was isolated, and showed many differences from the wild-type strain. A 3.6 kb DNA fragment that complemented the colony morphology defects of this mutant contained two open reading frames (ORFs). One ORF, designated ssfR, encoded a protein containing a potential DNA-bonding helix-turn-helix motif close to its N-terminus. SsfR is similar to a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (Kawamoto and Ensign, 1995). The ssfR null mutant showed the phenotypes of both bald and white mutants, and the ssgA disruption mutant had a white mutant phenotype. The phenotypes showed some medium dependence.