Genetic characterization of mycobacterial L,D-transpeptidases.

Akeisha N Sanders, Lori F Wright, Martin S Pavelka

Microbiology (Reading, England) 2014 Aug

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l,d-Transpeptidases (Ldts) catalyse the formation of 3-3 cross-links in peptidoglycans (PGs); however, the role of these enzymes in cell envelope physiology is not well understood. Mycobacterial PG contains a higher percentage of 3-3 cross-links (~30-80 %) than the PG in most other bacteria, suggesting that they are particularly important to mycobacterial cell wall biology. The genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis encode multiple Ldt genes, but it is not clear if they are redundant. We compared the sequences of the Ldt proteins from 18 mycobacterial genomes and found that they can be grouped into six classes. We then constructed M. smegmatis strains lacking single or multiple Ldt genes to determine the physiological consequence of the loss of these enzymes. We report that of the single mutants, only one, ΔldtC (MSMEG_0929, class 5), displayed an increased susceptibility to imipenem - a carbapenem antibiotic that inhibits the Ldt enzymes. The invariant cysteine in the active site of LdtC was required for function, consistent with its role as an Ldt. A triple mutant missing ldtC and both of the class 2 genes displayed hypersusceptibility to antibiotics, lysozyme and d-methionine, and had an altered cellular morphology. These data demonstrated that the distinct classes of mycobacterial Ldts may reflect different, non-redundant functions and that the class 5 Ldt was peculiar in that its loss, alone and with the class 2 proteins, had the most profound effect on phenotype. © 2014 The Authors.

Citation

Akeisha N Sanders, Lori F Wright, Martin S Pavelka. Genetic characterization of mycobacterial L,D-transpeptidases. Microbiology (Reading, England). 2014 Aug;160(Pt 8):1795-1806