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

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.

The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide

“…However, these binding interactions of the tripeptide species would probably be weaker than those of 1,6-anhydroMurNAc-pentapeptide or free pentapeptide that contain the D-Ala-D-Ala motif. Moreover, involvement of the D-Ala-D-Ala motif in AmpR regulation is entirely consistent with the observed AmpC hyperproduction that occurs upon loss of PBP4 (24) because this protein has a DD-peptidase activity that removes the terminal D-Ala residue from PG catabolites (26). Thus, our findings support a scenario where loss of PBP4 causes 1,6-anhydroMurNAc-pentapeptide (or free pentapeptide) to accumulate and drive AmpR to hyperproduce AmpC.…”

“…Loss of PBP4 has recently been found to be the leading cause of single-step mutational resistance to ␤-lactams in P. aeruginosa (25). Interestingly, PBP4 removes the terminal D-Ala residue from PG catabolites prior to being transported to the cytosol for recycling (26), suggesting that the terminal D-Ala residue may be important for AmpC activation. Although occurring less frequently than ampD or dacB null mutations, mutations in ampR have also been found to drive constitutive high level AmpC production (21,25,27,28).…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…Together with PBP5/DacA, PBP4/DacB is involved in maintaining normal cell morphology [105][106][107]. PBP5/DacA is found attached to the inner membrane [108].…”

“…coli has five LMM PBPs, namely PBP4/DacB, PBP5/DacA, PBP6/DacC, PBP6b/DacD and PBP7/8/PbpG [74,79,103,104] ( Table 2). The PBP4/DacB is a bifunctional peptidase with both carboxy and endopeptidase activities [105].…”

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography

“…Being penicillin-sensitive enzymes, they belong to the family of penicillin-binding proteins (PBPs). Whereas PBP 5 and PBP 6 are specific carboxypeptidases (29), PBP 4 has been found, in addition to its DD-carboxypeptidase activity, to hydrolyze the murein-cross-linking DD-peptide bond between D-Ala and m-A2pm, thus also showing endopeptidase activity (19).…”