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Genetic characterization of mycobacterial L,D-transpeptidases
Akeisha N Sanders1, Lori F Wright1, Martin S Pavelka1
1Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Abstract:
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.
Insights
Mycobacterial l,d-Transpeptidases (Ldts) have distinct, non-redundant roles in cell wall synthesis. Loss of LdtC and class 2 Ldts significantly impacts mycobacterial physiology and antibiotic susceptibility.
Area of Science:
- Microbiology
- Biochemistry
- Cell Biology
Background:
- l,d-Transpeptidases (Ldts) are enzymes that catalyze the formation of 3-3 cross-links in peptidoglycans (PGs).
- Mycobacterial PG has a high percentage of 3-3 cross-links, suggesting Ldts are crucial for mycobacterial cell wall integrity.
- The functional redundancy of multiple Ldt genes in mycobacteria remains unclear.
Purpose of the Study:
- To investigate the physiological roles and potential redundancy of l,d-Transpeptidase (Ldt) enzymes in mycobacteria.
- To determine the consequences of deleting single or multiple Ldt genes in Mycobacterium smegmatis.
Main Methods:
- Comparative sequence analysis of Ldt proteins across 18 mycobacterial genomes to establish classifications.
- Construction and phenotypic analysis of Mycobacterium smegmatis strains with single and multiple Ldt gene deletions.
Main Results:
- Single deletion of ldtC (class 5 Ldt) increased susceptibility to imipenem, an Ldt-inhibiting antibiotic.
- A triple mutant lacking ldtC and two class 2 Ldt genes exhibited hypersusceptibility to antibiotics, lysozyme, and D-methionine.
- The triple mutant also displayed significant alterations in cellular morphology.
Conclusions:
- Distinct classes of mycobacterial Ldts likely possess non-redundant functions.
- LdtC (class 5) plays a unique role, as its absence, alone or with class 2 Ldts, profoundly affects mycobacterial phenotype and antibiotic resistance.
