Characterization of a Mycobacterium smegmatis uvrA mutant impaired in dormancy induced by hypoxia and low carbon concentration

<p>Abstract</p> <p>Background</p> <p>The aerobic fast-growing <it>Mycobacterium smegmatis</it>, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of <it>M. smegmatis </it>dormancy, by identifying and characterizing genes involved in this behavior.</p> <p>Results</p> <p>In a library generated by transposon mutagenesis, we searched for <it>M. smegmatis </it>mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the <it>uvrA </it>gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the <it>uvrA </it>gene. The restoration of the <it>uvrA </it>activity in <it>M. smegmatis </it>by complementation with the <it>uvrA </it>gene of <it>M. tuberculosis</it>, confirmed that i) <it>uvrA </it>inactivation was indeed responsible for the inability of <it>M. smegmatis </it>cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective <it>uvrA </it>genes of <it>M. tuberculosis </it>and <it>M. smegmatis </it>are true orthologs. The rate of survival of wild type, <it>uvrA </it>mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively.</p> <p>Conclusions</p> <p>Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.</p

ICDS database: interrupted CoDing sequences in prokaryotic genomes

Unrecognized frameshifts, in-frame stop codons and sequencing errors lead to Interrupted CoDing Sequence (ICDS) that can seriously affect all subsequent steps of functional characterization, from in silico analysis to high-throughput proteomic projects. Here, we describe the Interrupted CoDing Sequence database containing ICDS detected by a similarity-based approach in 80 complete prokaryotic genomes. ICDS can be retrieved by species browsing or similarity searches via a web interface (). The definition of each interrupted gene is provided as well as the ICDS genomic localization with the surrounding sequence. Furthermore, to facilitate the experimental characterization of ICDS, we propose optimized primers for re-sequencing purposes. The database will be regularly updated with additional data from ongoing sequenced genomes. Our strategy has been validated by three independent tests: (i) ICDS prediction on a benchmark of artificially created frameshifts, (ii) comparison of predicted ICDS and results obtained from the comparison of the two genomic sequences of Bacillus licheniformis strain ATCC 14580 and (iii) re-sequencing of 25 predicted ICDS of the recently sequenced genome of Mycobacterium smegmatis. This allows us to estimate the specificity and sensitivity (95 and 82%, respectively) of our program and the efficiency of primer determination

Detecting the molecular scars of evolution in the Mycobacterium tuberculosis complex by analyzing interrupted coding sequences

<p>Abstract</p> <p>Background</p> <p>Computer-assisted analyses have shown that all bacterial genomes contain a small percentage of open reading frames with a frameshift or in-frame stop codon We report here a comparative analysis of these interrupted coding sequences (ICDSs) in six isolates of <it>M. tuberculosis</it>, two of <it>M. bovis </it>and one of <it>M. africanum </it>and question their phenotypic impact and evolutionary significance.</p> <p>Results</p> <p>ICDSs were classified as "common to all strains" or "strain-specific". Common ICDSs are believed to result from mutations acquired before the divergence of the species, whereas strain-specific ICDSs were acquired after this divergence. Comparative analyses of these ICDSs therefore define the molecular signature of a particular strain, phylogenetic lineage or species, which may be useful for inferring phenotypic traits such as virulence and molecular relationships. For instance, <it>in silico </it>analysis of the W-Beijing lineage of <it>M. tuberculosis</it>, an emergent family involved in several outbreaks, is readily distinguishable from other phyla by its smaller number of common ICDSs, including at least one known to be associated with virulence. Our observation was confirmed through the sequencing analysis of ICDSs in a panel of 21 clinical <it>M. tuberculosis </it>strains. This analysis further illustrates the divergence of the W-Beijing lineage from other phyla in terms of the number of full-length ORFs not containing a frameshift. We further show that ICDS formation is not associated with the presence of a mutated promoter, and suggest that promoter extinction is not the main cause of pseudogene formation.</p> <p>Conclusion</p> <p>The correlation between ICDSs, function and phenotypes could have important evolutionary implications. This study provides population geneticists with a list of targets, which could undergo selective pressure and thus alters relationships between the various lineages of <it>M. tuberculosis </it>strains and their host. This approach could be applied to any closely related bacterial strains or species for which several genome sequences are available.</p

Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae

<p>Abstract</p> <p>Background</p> <p>The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism <it>Mycobacterium smegmatis</it>, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb.</p> <p>Results</p> <p>We have recently sequenced the complete genomes of two fast-growers causing human infections, <it>Mycobacterium abscessus </it>(CIP 104536T) and <it>M. chelonae </it>(CIP 104535T). We show here that these two species contain genes corresponding to all those of the <it>M. smegmatis </it>"GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in <it>M. chelonae </it>and <it>M. abscessus</it>. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (<it>atf1 </it>and <it>atf2</it>) in <it>M. abscessus </it>and <it>M. chelonae </it>has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (<it>M. chelonae</it>, 900 kb; <it>M. abscessus</it>, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.</p> <p>Conclusion</p> <p>Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in <it>M. smegmatis </it>represents the ancestral form and that evolution has scattered various pieces throughout the genome in <it>M. abscessus </it>and <it>M. chelonae</it>.</p

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

Conditional Gene Expression in Mycobacterium abscessus

Mycobacterium abscessus is an emerging human pathogen responsible for lung infections, skin and soft-tissue infections and disseminated infections in immunocompromised patients. It may exist either as a smooth (S) or rough (R) morphotype, the latter being associated with increased pathogenicity in various models. Genetic tools for homologous recombination and conditional gene expression are desperately needed to allow the study of M. abscessus virulence. However, descriptions of knock-out (KO) mutants in M. abscessus are rare, with only one KO mutant from an S strain described so far. Moreover, of the three major tools developed for homologous recombination in mycobacteria, only the one based on expression of phage recombinases is working. Several conditional gene expression tools have recently been engineered for Mycobacterium tuberculosis and Mycobacterium smegmatis, but none have been tested yet in M. abscessus. Based on previous experience with genetic tools allowing homologous recombination and their failure in M. abscessus, we evaluated the potential interest of a conditional gene expression approach using a system derived from the two repressors system, TetR/PipOFF. After several steps necessary to adapt TetR/PipOFF for M. abscessus, we have shown the efficiency of this system for conditional expression of an essential mycobacterial gene, fadD32. Inhibition of fadD32 was demonstrated for both the S and R isotypes, with marginally better efficiency for the R isotype. Conditional gene expression using the dedicated TetR/PipOFF system vectors developed here is effective in S and R M. abscessus, and may constitute an interesting approach for future genetic studies in this pathogen

Lsr2 of Mycobacterium tuberculosis is a DNA-bridging protein

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2

Développement d'outils génétiques pour étudier la virulence de Mycobacterium abscessus

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Biosynthèse et export des glycopeptidolipides à la surface des mycobactéries

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF