Evaluation of NAD(+)-dependent DNA ligase of mycobacteria as a potential target for antibiotics

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

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.The study was supported by POIG.01.01.02-10-107/09 project implemented under Innovative Economy Operational Programme, years 2007–2013 "Studies of the molecular mechanisms at the interface the human organism - the pathogen - environmental factors" and by grant of Polish National Center of Science 2011/01/N/NZ6/04186 “Identification of a novel mechanism of initiation of DNA replication in Mycobacterium smegmatis”

Evaluation of DNA primase DnaG as a potential target for antibiotics

Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target

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

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway

Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli

The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5′-triphosphate. This fact leads to the conclusion that NAD+-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD+ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD+-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

<div><p>Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the <em>Mycobacterium smegmatis</em>, <em>Mycobacterium tuberculosis</em> and <em>Escherichia coli</em> genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in <em>M. smegmatis</em>, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the <em>attB</em> integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.</p> </div

Southern blot confirming deletion of native <i>dnaA</i> gene in ∆<i>rnhA</i>/∆<i>dnaAattB</i>::<i>dnaA</i> mutant strain of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in <i>rnhA</i>. Further, we used the same procedure to generate ∆<i>rnhA</i>/<i>dnaA</i>SCO strain where SCO signifies an intermediate step of gene replacement procedure. Next, through complementation procedure, we introduced an additional version of <i>dnaA</i> gene at the <i>attB</i> site of mycobacterial genome. Finally, we removed the native version of <i>dnaA</i>, thereby generating a mutant ∆<i>rnhA</i>/<i>dnaA</i>SCO<i>attB</i>::<i>dnaA</i>. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic region, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic film presenting results of Southern blot analysis is presented in the lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of the photograph.</p

Southern blots confirming deletions in single RNase H type I mutants of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in either <i>rnhA</i> or MSMEG4305. Briefly, recombinant plasmids containing genomic regions of either <i>rnhA</i> or MSMEG4305 with large deletions within each gene were introduced into <i>M</i>. <i>smegmatis</i> mc² 155 cells. Following multistep selection we were able to identify clones where the native version of each gene has been replaced with manipulated sequence. Intermediate steps of gene replacement procedure are denoted SCO. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic regions, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic films presenting results of Southern blot analysis are presented in lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of each photograph.</p

Primers used in this study.

<p>Primers used in this study.</p

Comparison of protein sequences of RNases H type I.

<p>Sequence alignment between RNases H type I of <i>E</i>. <i>coli</i> K12_MG1655 (RnhA), <i>M</i>. <i>smegmatis</i> mc² 155 (MSMEG4305 and RnhA) and <i>M</i>. <i>tuberculosis</i> H37Rv (Rv2228c) was performed using MultiAlin and visualized with ESPript 3.0. Highly similar or identical residues between protein sequences are written in bold. Identical residues across all analyzed sequences are shown in white on a black background. Similarities between protein sequences are marked by framing. The span of RNase H domains in each protein sequence, as defined by SMART, is highlighted in yellow.</p