LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most ß-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

Genetic characterization of mycobacterial L,D-transpeptidases.

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.

Phenotypic analysis of Eschericia coli mutants lacking L,D-transpeptidases.

Escherichia coli has five genes encoding L,D-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun's lipoprotein to the peptidoglycan (PG), while the other two (YnhG, YcbB) form direct meso-diaminopimelate (DAP-DAP, or 3-3) cross-links within the PG. In addition, Ldt enzymes can also incorporate non-canonical D-amino acids, such as D-methionine, into the PG. To further investigate the role of these enzymes and, in particular, 3-3 linkages in cell envelope physiology we constructed and phenotypically characterized a variety of multiple Ldt deletion mutants of E. coli. We report that a triple deletion mutant lacking ybiS, erfK and ycfS is hypersusceptible to the metal-chelating agent EDTA, leaks periplasmic proteins and is resistant to the toxic effect of D-methionine. A double ynhG ycbB mutant had no discernible phenotype; however, examination of the phenotypes of various Ldt mutants bearing an additional DAP auxotrophic mutation (dapA : : Cm) showed that a quintuple mutant strain lacking all Ldt genes was severely impaired for growth on media with limited DAP. These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of outer membrane stability while loss of the Ldt enzymes involved with DAP-DAP linkages had no observable effect on the cell envelope. Loss of all Ldt enzymes proved detrimental to growth when cells were starved for DAP, indicating a combined role for both 3-3 and Braun's lipoprotein cross-links in cell viability only under a specific PG stress.

Normal regulation of Rbf1/E2f1 target genes in Drosophila type 1 protein phosphatase mutants.

G1 Cyclin/Cdk complexes phosphorylate and inactivate the pRb tumor suppressor by preventing its ability to bind and repress E2F transcription factors. Current molecular and biochemical evidence suggests that type 1 protein phosphatases (PP1) dephosphorylate and thereby activate pRb, but the functional significance of this has not been addressed in the context of animal development. Here, we use genetic analyses to determine the role of PP1 in the regulation of Rbf1 activity during Drosophila development. While Rbf1 is required for E2f1 inhibition and G1 arrest in the embryonic epidermis and for the periodic expression of E2f1 target genes during endocycle S phase in the embryonic midgut and larval salivary gland, PP1 is not. PP1 regulates periodic cyclin E protein accumulation in ovarian nurse cells independently of Rbf1, which is dispensable for endocycle regulation in this tissue. We conclude that PP1 is not a major regulator of the Rbf1/E2F1 pathway in Drosophila.