Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Korycka-Machała, Małgorzata; Brzostek, Anna; Różalska, Sylwia; Rumijowska-Galewicz, Anna; Dziedzic, Renata; Bowater, Richard P.; Dziadek, Jarosław

doi:10.1111/j.1574-6968.2006.00199.x

Cited by 25 publications

(23 citation statements)

References 32 publications

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“…qRT-PCR analysis of all accD members in M. tuberculosis and M. smegmatis showed that the three accD genes highly expressed and regulated during mycolate biosynthesis in pathogenic mycobacteria were (11,12,13,20,29,30). In contrast to procedures based on delivery vectors harboring a thermosensitive origin of replication (25,56), this method does not require growth temperature shifts (54).…”

Section: Discussionmentioning

confidence: 99%

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

et al. 2011

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Acetyl coenzyme A carboxylase (ACC) is a key enzyme providing a substrate for mycolic acid biosynthesis. Although in vitro studies have demonstrated that the protein encoded by accD6 (Rv2247) may be a functional carboxyltransferase subunit of ACC in Mycobacterium tuberculosis, the in vivo function and regulation of accD6 in slow-and fast-growing mycobacteria remain elusive. Here, directed mutagenesis demonstrated that although accD6 is essential for M. tuberculosis, it can be deleted in Mycobacterium smegmatis without affecting its cell envelope integrity. Moreover, we showed that although it is part of the type II fatty acid synthase operon, the accD6 gene of M. tuberculosis, but not that of M. smegmatis, possesses its own additional promoter (P acc ). The expression level of accD6 Mtb placed only under the control of P acc is 10-fold lower than that in wild-type M. tuberculosis but is sufficient to sustain cell viability. Importantly, this limited expression level affects growth, mycolic acid content, and cell morphology. These results provide the first in vivo evidence for AccD6 as a key player in the mycolate biosynthesis of M. tuberculosis, implicating AccD6 as the essential ACC subunit in pathogenic mycobacteria and an excellent target for new antitubercular compounds. Our findings also highlight important differences in the mechanism of acetyl carboxylation between pathogenic and nonpathogenic mycobacterial species.

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Section: Discussionmentioning

confidence: 99%

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

et al. 2011

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“…NHEJ-deficient M. smegmatis strains used in this work were as previously described by Korycka-Machala et al (5).…”

Section: Bacterial Strainsmentioning

confidence: 99%

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

Pitcher¹,

Green²,

Brzostek³

et al. 2007

DNA Repair

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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.

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“…Following strategies reported previously that deleted DNA ligase genes in M. smegmatis (12), a suicidal recombination delivery vector was constructed to perform unmarked deletions in the ligA gene (MSMEG2361) of M. smegmatis. The vector carried the 5Ј end of ligA (68 bp) with the upstream region amplified with primers A-GR1 and A-GR2 (Table 2) connected to the 3Ј end of the gene (936 bp) and with the downstream region amplified with A-GR3 and A-GR4 primers ( (Tables 1 and 2).…”

Section: Methodsmentioning

confidence: 99%

“…A two-step recombination protocol (12,17) was used to obtain single-crossover (SCO) strains carrying wild-type (wt) and disrupted ligA (⌬ligA) at its native locus on the chromosome. Using the strategy outlined in Fig.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of NAD ⁺ -Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

Korycka-Machała

Rychta

Brzostek

et al. 2007

Antimicrob Agents Chemother

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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...

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Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Cited by 25 publications

References 32 publications

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

Evaluation of NAD ⁺ -Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

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Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Cited by 25 publications

References 32 publications

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

Evaluation of NAD + -Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

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Evaluation of NAD ⁺ -Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics