Abstract
BackgroundBacterial 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).ResultsEight 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.ConclusionsNine 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.
Highlights
Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent b-lactam derivatives both whole cells and membrane/cell enriched fractions
Examination of sequence information from a database dedicated to the analysis of the genomes of L. monocytogenes and its non-pathogenic relative Listeria innocua http:// genolist.pasteur.fr/ListiList, as well as that from the Pfam database http://www.sanger.ac.uk/Software/Pfam and information from the NCBI Conserved Domain database http://www.ncbi.nlm.nih.gov/COG/ and the Interpro database http://www.ebi.ac.uk/interpro/, has identified 10 putative genes for PBPs, classified according to molecular class (Table 1)
The inability to detect Lmo2812 activity in the L. monocytogenes cell may be explained by the low abundance of this protein, whose expression is regulated by the two-component system CesRK [21]
Summary
Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent b-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, hindering their characterization. Penicillin-binding proteins (PBPs) are responsible for the final synthesis steps of the universal peptidoglycan exoskeleton of bacteria. Since their initial identification by Brian Spratt [1] most attention has been paid to the activities of these proteins in model microorganisms such as Escherichia coli, Bacillus subtilis and Streptococcus pneumoniae. Earlier studies carried out in our laboratory - when only five PBPs were known - resulted in a re-estimation of the copy number of individual L. monocytogenes penicillin-binding proteins [10] and elucidation of the enzymatic properties of PBP4 (encoded by lmo2229) and PBP5 (lmo2754) [11,12,13]. The insertional mutagenesis of genes encoding seven potential PBPs -two of class A, three of the high molecular mass (HMM) class B and two of the low molecular mass (LMM) type - helped to clarify their role [15]
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