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Summary: Natural modification of the colony appearance is a phenomenon that has not been fully understood in mycobacteria. Here, we show that Mycobacterium smegmatis ATCC607 displays a low-frequency spontaneous morphological variation that correlates with the acquisition of a panel of new phenotypes, such as aggregation, biofilm formation and sliding motility. These variants produce larger amounts of glycopeptidolipid (GPL), a cell-surface component, than did the wild-type strain. This conversion results from the transposition of two types of insertion sequences, IS1096 and ISMsm3, into two loci. One locus is the promoter region of the mps operon, the GPL biosynthesis gene cluster, leading to the overexpression of these genes. The other locus is the lsr2 gene, which encodes a small basic histone-like protein that likely plays a regulatory role at the mps promoter and also controls pigment production. This study demonstrates that insertion sequence mobility play a crucial role in the acquisition of new phenotypes. [Copyright &y& Elsevier]

The EMBO–FEBS lecture course on the ‘Molecular Basis of Bacterial Virulence and Survival Within Infected Hosts and in the Environment’, organized by Pascale Cossart, Efstathios Gonos and Roberto Kolter, was held on the island of Spetsai, Greece, September 3–13, 2002. [Copyright &y& Elsevier]

Mycobacterium abscessus, a relative of Koch''s bacillus (the bacterium that causes tuberculosis), has recently emerged as the cause of an increasing number of both community- and hospital-acquired infections in humans; it also constitutes a serious threat for cystic fibrosis patients. This situation is worsened by its exceptionally high natural and acquired antibiotic resistance that complicates treatment. Although a rapid grower, it shares some traits with Koch''s bacillus, including the ability to induce a persistent lung disease associated with caseous lesions, a landmark of Mycobacterium tuberculosis infection. Its genome sequence and microarrays are now available, and efficient genetic tools have recently been developed. Here we consider the various advantages of using this species as an experimental model to study tuberculosis and other related mycobacterial diseases. [Copyright &y& Elsevier]

Mycobacterium smegmatis is a saprophytic species that has been used for 15 years as a model to perform heterologous regulation and virulence studies of Mycobacterium tuberculosis. Members of the extracytoplasmic sigma factors family, which are required for adaptive responses to various environmental stresses, are responsible for some of the virulence traits of M. tuberculosis. A bioinformatic search on the genome of M. smegmatis has predicted the existence of 26 sigma factors, which is twice the number that are present in M. tuberculosis. A phylogenetic analysis has shown that despite this high number of sigma factors the orthologs of the genes sigC, sigI and sigK of M. tuberculosis are absent in the M. smegmatis genome. Several sigma factors are specific for M. smegmatis, with a special enrichment in the sigH and, to a lesser extent, in the sigJ and sigL subfamily, pinpointing the potential variability of the repertoire of adaptive response in this saprophytic species. [Copyright &y& Elsevier]

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe- Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl- AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. [ABSTRACT FROM AUTHOR]

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnbsyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-D-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex. [ABSTRACT FROM AUTHOR]

Summary: Background: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne''s disease in cattle, and several conflicting data have been produced. Methods: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals. Conclusions: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]