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

Mol Microbiol. 1991 Mar;5(3):675-84. doi: 10.1111/j.1365-2958.1991.tb00739.x.


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.

MeSH terms

  • 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


  • Bacterial Proteins
  • Carrier Proteins
  • Escherichia coli Proteins
  • Penicillin-Binding Proteins
  • Peptidoglycan
  • Peptidyl Transferases
  • Hexosyltransferases
  • Serine-Type D-Ala-D-Ala Carboxypeptidase
  • penicillin-binding protein 4, E coli
  • Muramoylpentapeptide Carboxypeptidase