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Background: The aerobic fast-growing Mycobacterium smegmatis, 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 M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior., Results: In a library generated by transposon mutagenesis, we searched for M. smegmatis 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 uvrA 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 uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively., Conclusions: 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.

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery., (© 2010 Blackwell Publishing Ltd.)

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that sigma(F) is required for carotenoid pigment production in Mycobacterium smegmatis. We further show that a sigF mutant exhibits a transformation efficiency 10(4)-fold higher than that of the parental strain, suggesting that sigma(F) regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.

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.

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

Erp (exported repetitive protein) is a member of a mycobacterium-specific family of extracellular proteins. A hydrophobic region that is localized at the C-terminal domain and that represents a quarter of the protein is highly conserved across species. Here we show that this hydrophobic region is not essential for restoring the virulence and tissue damage of an erp::aph mutant strain of M. tuberculosis as assessed by bacterial counts and lung histology analysis in a mouse model of tuberculosis.

Erp (Exported Repetitive Protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria-specific family of extracellular proteins. In pathogenic species, the erp gene has been described as a virulence factor. The Erp proteins comprise three domains. The N- and C-terminal domains are similar in all mycobacterial species, while the central domain consists of a repeated module that differs considerably between species. Here we show that the Erp protein is loosely attached to the surface and that the carboxy-terminal domain, which displays hydrophobic features, anchors Erp at the surface of the bacillus. The hydrophobic region is not necessary for the complementation of the altered colony morphology of a Mycobacterium smegmatis erp- mutant but proved to be necessary to achieve resistance to detergent at wild-type levels.

Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing C-terminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.

Erp (exported repetitive protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria-specific family of extracellular proteins. These proteins consist of three domains, the N- and C-terminal domains are similar in all mycobacterial species, however, the central domain contains a repeated PGLTS module and differs considerably between species. The erp knockout mutant of Mycobacterium tuberculosis displays very low levels of multiplication both in macrophage cell lines and in vivo in a mouse model of infection. The high interspecies variability of the central repeated region of the Erp protein led us to investigate whether these orthologous proteins were functionally equivalent in a mouse model of tuberculosis. We expressed a gene fusion with the erp gene of Mycobacterium smegmatis, Mycobacterium leprae or M. tuberculosis in trans in an erp-M. tuberculosis mutant and found that these three alleles restored multiplication to similar levels in the spleen of infected mice. However, these alleles gave different levels of colonization in the lung, for the early time-points. Quantitative histological analyses of the lungs of infected animals showed that the nature of the erp allele strongly affected the number and the size of lung lesions, demonstrating the importance of surface determinants for virulence and tissue damage.

Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.

There are two functionally different alleles of the Helicobacter pylori vacA gene, which code for proteins with different in target cell specificity. The alleles (m1 and m2) differ by approximately 50% in amino acid sequence in a 300 amino acid region, the m-region, which determines specificity. An analysis of partial likelihood anomalies in a set of eight Chinese and six Western vacA genes revealed highly significant phylogenetic deviation of a region of the gene including the m-region. Phylogenetic analysis of the conserved regions of these genes failed to reveal any distinction between m1 alleles and m2 alleles, however clear cut geographic variation was observed. In the m-region, the m1 alleles also show separate clustering of Chinese and Western isolates, however the m-region of the m2 alleles has a phylogenetic structure markedly different from the rest of the gene. The data indicate that the m2 m-region was acquired and spread through the population by horizontal transfer of DNA.

Erp (exported repeated protein) was originally characterized as a virulence factor in Mycobacterium tuberculosis and was thought to be present only in Mycobacterium leprae and members of the TB complex. Here it is shown that Erp is a ubiquitous extracellular protein found in all of the mycobacterial species tested. Erp proteins have a modular organization and contain three domains: a highly conserved amino-terminal domain which includes a signal sequence, a central variable region containing repeats based on the motif PGLTS, and a conserved carboxy-terminal domain rich in proline and alanine. The number and fidelity of PGLTS repeats of the central region differ considerably between mycobacterial species. This region is, however, identical in all of the clinical M. tuberculosis strains tested. In addition, it is shown here that a Mycobacterium smegmatis erp::aph mutant displays altered colony morphology which is complemented by all the Erp orthologues tested. The genome sequence flanking the erp gene includes cell-wall-related ORFs and displays extensive conservation between saprophytic and pathogenic mycobacteria.

Polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells were used to study the pathogenicity of Helicobater pylori, with an emphasis on the effect of VacA. The adherence of H. pylori to MDCK monolayers resulted in a decrease in trans-epithelial resistance (TER) across the cell monolayer. Isogenic vacA mutants did not lower the TER, demonstrating that the effect is strictly linked to the action of the toxin. A similar effect was observed with all VacA-producing strains, including those producing m2 toxins that are inactive in the vacuolating assay. In contrast to that seen with purified toxin, TER decrease was not enhanced by acid pH, which may indicate that the toxin associated to the bacterial surface is possibly in a monomeric state and therefore does not require a pH-induced conformation to be active. These data raise the possibility that one role of VacA in ulcerogenesis may consist of increasing the paracellular permeability of the gastric epithelium.