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Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition

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Korat,  B
Department Biochemistry, Max Planck Institute for Developmental Biology, Max Planck Society;

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Citation

Korat, B., Mottl, H., & Keck, W. (1991). Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition. Molecular Microbiology, 5(3), 675-684. doi:10.1111/j.1365-2958.1991.tb00739.x.


Cite as: https://hdl.handle.net/21.11116/0000-000F-1EE2-1
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.