Genetic characterization of mycobacterial L,D-transpeptidases

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.

Fig. 1.

Structural classification of mycobacterial Ldts (Msm, M. smegmatis; Mtb, M. tuberculosis). Schematic protein organization of each class of mycobacterial Ldts is shown, derived from analyses of the translated sequences of the Ldt genes in 18 mycobacterial genomes. The proteins can be grouped into six classes. Class 5 and 2 are lipoproteins (Lipobox) and also contain a PRR C terminus. Class 1 proteins contain a conserved Region 1 sequence, whilst class 3 and class 6 share a conserved Region 2 sequence along with the Region 1 sequence. Class 6 proteins also have an internal PRR sequence. Class 4 proteins have a conserved PRR sequence in the N terminus. Black boxes at the N termini of some of the proteins indicate predicted transmembrane helices according to genome annotation. All classes contain the characteristic active-site region, HXX_14–17[S/T]HGChN (where h is a hydrophobic residue), containing the catalytic cysteine residues. Note that two residues preceding the cysteine, only class 5 has an asparagine instead of the conserved histidine.

Fig. 2.

Lysozyme sensitivity of the class 5 and class 2 triple-mutant strain. WT and mutant strains were grown to the mid-exponential phase of growth and plated for viable cell counts on either 7H10 medium or 7H10 supplemented with 0.2 mg lysozyme (Lys) ml⁻¹. (a) Class 5 (PM2110) and class 2 (PM2232, PM2239) single-mutant strains, class 2 double-mutant strain (PM2269), and class 5 and 2 triple-mutant strain (PM2546). (b) Class 5 and class 2 triple-mutant strain was complemented with either pMV261 [vector control (VC)], or pMV261 containing either a class 5 or class 2 gene from M. smegmatis (Msm) or M. tuberculosis (Mtb) Data were analysed using Student’s t-test. All values were significant comparing lysozyme-treated and untreated samples for the same strain (P<0.01), and comparing lysozyme treated strains with the lysozyme-treated WT control (P<0.001).

Fig. 3.

d-Methionine sensitivity of the class 5 and class 2 triple-mutant strain. WT and mutant strains were grown to the mid-exponential phase of growth and plated for viable cell counts on either 7H10 medium or 7H10 supplemented with 15 mM d-methionine. Data were analysed using Student’s t-test, comparing d-methionine-treated and untreated samples for the same strain (P<0.001) *.

Fig. 4.

Cellular morphology of the class 5 and class 2 triple-mutant strain. Fluorescence microscopy of stationary-phase M. smegmatis strains expressing gfp from plasmid pMN437. (a) PM3070 (WT), (b) PM3071 (ΔldtC) and (c) PM3072 (ΔldtB, ΔldtF, ΔldtC).

Fig. 5.

RT-PCR of select ldt genes, performed on RNA purified from exponential-phase cell cultures. Lane 1, DNA size markers (bp); lanes 2 and 3, MSMEG_0223, class 4 (ldtE), 295 bp; lanes 4 and 5, MSMEG_0674, class 6 (ldtG), 248 bp; lanes 6 and 7,: MSMEG_3528, class 1, (ldtA) 278 bp. Reactions in lanes 2, 4 and 6 included reverse transcriptase, whilst the control reactions in lanes 3, 5 and 7 lacked the enzyme.