Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition.
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.
Korat
B
B
Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.
Mottl
H
H
Keck
W
W
D001426:Bacterial Proteins; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000096:biosynthesis / Q000737:chemistry*; D010458:Peptidyl Transferases; D010957:Plasmids; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C478706:penicillin-binding protein 4, E coli; D002267:Muramoylpentapeptide Carboxypeptidase