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Background  Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent β-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the  Listeria monocytogenes  genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studies using radiolabeled antibiotics, thus hindering their characterization. Here we report the positive identification of the full set of  L. monocytogenes  PBPs and the characteristics of the hitherto undescribed PBPD2 (Lmo2812).    Results  Eight  L. monocytogenes  PBPs were identified by the binding of fluorescent β-lactam antibiotic derivatives Boc-FL, Boc-650 and Amp-Alexa430 to proteins in whole cells or membrane/cell wall extracts. The gene encoding a ninth PBP (Lmo2812) was cloned and expressed in  Escherichia coli  as a His-tagged protein. The affinity purified recombinant protein had DD-carboxypeptidase activity and preferentially degraded low-molecular-weight substrates.  L. monocytogenes  mutants lacking the functional Lmo2812 enzyme were constructed and, compared to the wild-type, the cells were longer and slightly curved with bent ends.  Protein Lmo1855, previously designated PBPD3, did not bind any of the antibiotic derivatives tested, similarly to the homologous enterococcal protein VanY.    Conclusions  Nine out of the ten putative  L. monocytogenes  PBP genes were shown to encode proteins that bind derivatives of β-lactam antibiotics, thus enabling their positive identification. PBPD2 (Lmo2812) was not visualized in whole cell extracts, most probably due to its low abundance, but it was shown to bind Boc-FL after recombinant overexpression and purification. Mutants lacking Lmo2812 and another low molecular mass (LMM) PBP, PBP5 (PBPD1) - both with DD-carboxypeptidase activity - displayed only slight morphological alterations, demonstrating that they are dispensable for cell survival and probably participate in the latter stages of peptidoglycan synthesis. Since Lmo2812 preferentially degrades low-molecular- mass substrates, this may indicate a role in cell wall turnover.

Identification of the full set of Listeria monocytogenes penicillin-binding proteins and characterization of PBPD2 (Lmo2812)