Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years.
In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. Pharmaceutical Microbiology Section, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germanyįor intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far.It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.Stefania De Benedetti ‡, Henrike Bühl ‡, Ahmed Gaballah †, Anna Klöckner, Christian Otten, Tanja Schneider, Hans-Georg Sahl and Beate Henrichfreise * The results provide extremely strong evidence that the inactivation of PBP3 at 42☌ in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. Revertants that grew as normal rods at 42☌ regained both the normal level and the normal thermostability of PBP3. PBP3 was not stabilized in vitro at 42☌ by these concentrations of sucrose or MgCl2. The inhibition of division at 42☌ was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl2. At 42☌ division slowly recommenced, but at 44☌ this did not occur. At 30☌ both mutants were slightly longer than their parents and on shifting to 42☌ they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. The other PBPs were normal in both mutants. Binding of benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30☌, and the ability to bind benzylpenicillin was rapidly lost on incubation at 42☌.
Two independent cell division mutants have been found that have highly thermolabile PBP3. The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. SPRATT BG, 1977, Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins, Journal of Bacteriology, Vol: 131, Pages: 293-305, ISSN: 1098-5530
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