PMID:2046551
| Citation |
Korat, B, Mottl, H and Keck, W (1991) Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition. Mol. Microbiol. 5:675-84 |
|---|---|
| Abstract |
The penicillin-binding protein 4 (PBP4), from Escherichia coli, a DD-carboxypeptidase/DD-endopeptidase, was purified in an enzymatically active form to homogeneity by affinity chromatography on 6-aminopenicillanic acid/Sepharose and heparin/Sepharose. Polyclonal antibodies raised against the pure protein were used to identify and isolate PBP4 overproducing clones from an E. coli expression library, which was established on the basis of a temperature-inducible runaway replication plasmid. Three positive clones were isolated, one of which carried the intact structural gene dacB that codes for PBP4, on a 1.9kb SmaI-EcoRI fragment, whereas the other two carried truncated forms of this gene. The direction of transcription was determined. The PBP4 overproducing strain, when grown in rich medium, tolerated 160-fold overexpression. After disrupting cells by sonication, the majority (80%) of the overproduced PBP4 was detected in the 100,000 X g supernatant. Southern blotting analysis using the cloned dacB gene as a probe revealed that, in contrast to that described by Takeda et al. (1981), the plasmid pLC18-38 of the Clarke-Carbon collection does not code for PBP4. The overall composition of murein, synthesized in vitro or in vivo by the PBP4 overproducing strain, as determined by high-performance liquid chromatography analysis, suggests that PBP4 is not involved in transpeptidation but exclusively catalyses a DD-carboxypeptidase and DD-endopeptidase reaction. |
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| Keywords |
Bacterial Proteins; Blotting, Southern; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Chromosome Mapping; Chromosomes, Bacterial; Cloning, Molecular; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hexosyltransferases; Muramoylpentapeptide Carboxypeptidase/genetics; Muramoylpentapeptide Carboxypeptidase/isolation & purification; Muramoylpentapeptide Carboxypeptidase/metabolism; Penicillin-Binding Proteins; Peptidoglycan/biosynthesis; Peptidoglycan/chemistry; Peptidyl Transferases; Plasmids; Serine-Type D-Ala-D-Ala Carboxypeptidase; Solubility |
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