A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

Background: Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). Results: In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion: An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNA(ala )T-loops in slow- and fast-growing mycobacteria

BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA(ala )genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA(ala )loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA(ala )genes. RESULTS: The three tRNA(ala )genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNA(alaU )and tRNA(alaV )containing the attB site, a single base difference was observed between the two species. We observed that the tRNA(alaU )gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNA(alaV )gene was used as the target. No integration occurred in the BCG tRNA(alaU )T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNA(ala )T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNA(alaU )and tRNA(alaV )T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNA(ala )genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies

Multidrug-resistant Mycobacterium tuberculosis, Bangui, Central African Republic

We investigated multidrug-resistant (MDR) Mycobacterium tuberculosis strains in Bangui, Central African Republic. We found 39.6% with the same spoligotype and synonymous single nucleotide polymorphism in the mutT1 gene. However, strains had different rpoB mutations responsible for rifampin resistance. MDR strains in Bangui may emerge preferentially from a single, MDR-prone family

Phylogeny of Mycobacterium tuberculosis Beijing Strains Constructed from Polymorphisms in Genes Involved in DNA Replication, Recombination and Repair

The original publication is available at http:/www.plosone.orgBackground: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. Methodology/Principal Findings: A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant. Conclusions/Significance: We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family. © 2011 Mestre et al.Publishers' Versio

Evolution and Diversity of Clonal Bacteria: The Paradigm of Mycobacterium tuberculosis

International audienceBACKGROUND: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. PRINCIPAL FINDINGS: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. CONCLUSIONS/SIGNIFICANCE: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria

Evolution et diversité de Mycobacterium tuberculosis comme dit par les gardes de la stabilité du génome

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Dendrogram of the various spoligopatterns of strains harbouring variations at codon 337 or 337 and 79

<p><b>Copyright information:</b></p><p>Taken from "A non-sense mutation in the putative anti-mutator gene of and isolates suggests convergent evolution"</p><p>http://www.biomedcentral.com/1471-2180/7/39</p><p>BMC Microbiology 2007;7():39-39.</p><p>Published online 16 May 2007</p><p>PMCID:PMC1891112.</p><p></p

Isoxyl Activation Is Required for Bacteriostatic Activity against Mycobacterium tuberculosis▿

Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis

Different antibiotic resistance and sporulation properties within multiclonal Clostridium difficile PCR ribotypes 078, 126, and 033 in a single calf farm

Clostridium difficile strains were sampled periodically from 50 animals at a single veal calf farm over a period of 6 months. At arrival, 10% of animals were C. difficile positive, and the peak incidence was determined to occur at the age of 18 days (16%). The prevalence then decreased, and at slaughter, C. difficile could not be isolated. Six different PCR ribotypes were detected, and strains within a single PCR ribotype could be differentiated further by pulsed-field gel electrophoresis (PFGE). The PCR ribotype diversity was high up to the animal age of 18 days, but at later sampling points, PCR ribotype 078 and the highly related PCR ribotype 126 predominated. Resistance to tetracycline, doxycycline, and erythromycin was detected, while all strains were susceptible to amoxicillin and metronidazole. Multiple variations of the resistance gene tet(M) were present at the same sampling point, and these changed over time. We have shown that PCR ribotypes often associated with cattle (ribotypes 078, 126, and 033) were not clonal but differed in PFGE type, sporulation properties, antibiotic sensitivities, and tetracycline resistance determinants, suggesting that multiple strains of the same PCR ribotype infected the calves and that calves were likely to be infected prior to arrival at the farm. Importantly, strains isolated at later time points were more likely to be resistant to tetracycline and erythromycin and showed higher early sporulation efficiencies in vitro, suggesting that these two properties converge to promote the persistence of C. difficile in the environment or in hosts