Abstract:The physiological role of the non homologous end-joining (NHEJ) pathway in the repair of DNA double strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (∆recA, ∆ku, ∆ligD, ∆ku/ligD, ∆recA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ mutant strains (∆ku, ∆ligD, ∆ku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis.However, there was little difference in IR sensitivity between NHEJ-mutant and wildtype strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A ∆recA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the ∆recA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively.

The catabolic potential for sterol degradation of fast-growing mycobacteria is well known. However, no genes or enzymes responsible for the steroid degradation process have been identified as yet in these species. One of the key enzymes required for degradation of the steroid ring structure is 3-ketosteroid D 1 -dehydrogenase (KsdD). The recent annotation of the Mycobacterium smegmatis genome (TIGR database) revealed six KsdD homologues. Targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, resulted in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione. This effect was reversible by the introduction of the wild-type ksdD-1 gene into M. smegmatis DksdD-1 or overexpression of ksdD-2. The data indicate that KsdD1 is the main KsdD in M. smegmatis, but that KsdD2 is able to perform the cholesterol degradation process when overproduced.

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD ؉ -dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD ؉ -dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD ؉ -dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD ؉ -dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.Tuberculosis (TB) remains a major threat to public health. It is an important infectious disease, causing high morbidity and mortality worldwide, and the situation is made even worse by the emergence of drug-resistant strains of Mycobacterium tuberculosis (4). For example, multidrug-resistant (MDR) TB (resistant at least to rifampin and isoniazid) takes longer to treat (up to 2 years) with second-line drugs, which are expensive and have side effects. Even worse is extensively drugresistant TB, which is resistant to first-and second-line drugs (MDR plus resistance to any fluoroquinolone and at least one of three injectable second-line drugs: capreomycin, kanamycin, or amikacin). Thus, options for treatment are becoming seriously limited, returning TB control to the preantibiotic era (3,20). Drug resistance in M. tuberculosis is not caused by a universal mechanism for all drugs but can be caused by mutations of various chromosomal genes, as identified for MDR occurrence due to the sequential accumulation of mutations in different genes that provide resistance to individual drugs. The mutations connected to resistance can appear in targets of current drugs (e.g., inhA and kasA for isoniazid, rpoB for rifampin, and embCAB for ethambutol) or enzymes required for the intracellular activation o...

We have recently found that selected thio-disaccharides possess bactericidal effects against Mycobacterium tuberculosis but not against Escherichia coli or Staphylococcus aureus. Here, we selected spontaneous mutants displaying resistance against the investigated thio-glycoside. According to next-generation sequencing, four of six analyzed mutants which were resistant to high concentrations of the tested chemical carried nonsynonymous mutations in the gene encoding the PPE51 protein. The complementation of these mutants with an intact ppe51 gene returned their sensitivity to the wild-type level. The uptake of tritiated thio-glycoside was significantly more abundant in wild-type Mycobacterium tuberculosis compared to the strain carrying the mutated ppe51 gene. The ppe51 mutations or CRISPR-Cas9-mediated downregulation of PPE51 expression affected the growth of mutant strains on minimal media supplemented with disaccharides (maltose or lactose) but not with glycerol or glucose as the sole carbon and energy source. Taking the above into account, we postulate that PPE51 participates in the uptake of disaccharides by tubercle bacilli.