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

BMC Microbiology

Research Article

Juan A Ayala¹  Gabriel O Gutkind²  Dorota Korsak³  Zdzislaw Markiewicz³ 

[1] Centro de Biologia Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas, CSIC-UAM, C/Nicolás Cabrera 1, 28049, Madrid, Spain;Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina;Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland;

关键词: High Performance Liquid Chromatography;    Peptidoglycan;    Nitrocefin;    Double Mutant Strain;    Apparent Affinity Constant;   

DOI  :  10.1186/1471-2180-10-239

 received in 2010-03-30, accepted in 2010-09-15,  发布年份 2010

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Korsak et al; licensee BioMed Central Ltd. 2010