As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.Mycobacterium tuberculosis-the bacterium responsible for almost 2 million human deaths each year due to tuberculosis (TB)-exhibits a highly clonal population structure and, unlike many pathogenic bacteria, shows little evidence of having recently acquired exogenous DNA from unrelated organisms via horizontal gene transfer and recombination (30). Thus, it seems that the primary mechanism by which M. tuberculosis is able to modify existing phenotypes is through an alteration in the complement of genetic material that it already has available. To date, most of the phenotypic variation that has been reported for M. tuberculosis involves single nucleotide polymorphisms (SNPs) and deletions, although transposition of conserved insertion sequence elements (e.g., IS6110) represents another potential mode for generating variability (1,14,16,22). Recent SNP-and microarray-based whole-genome surveys have demonstrated that M. tuberculosis has evolved via clonal expansion into six major lineages that are referred to colloquially as Indo-Oceanic, East Asian, East African-Indian, Euro-American, West African-1, and West African-2. Aside from the widespread Euro-American lineage, each of these lineages tends to exhibit a strong degree of association with a particular geographic area (19,29,38).Of all the major lineages described to date, it is...

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bIn the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of the Mycobacterium tuberculosis East Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired for in vivo growth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of hostapplied selective pressure ( , here we show that this large chromosomal amplification event is very rapidly selected within standard in vitro broth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds of in vitro passage. In contrast, the duplication appears to be highly unstable in vivo and is negatively selected during the later stages of infection in mice. We believe that the rapid in vitro evolution of M. tuberculosis is an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.

Cholesterol has been reported to play an important role during Mycobacterium tuberculosis infection and during its dormant state inside the host. We present the determination of proteomic profiles of M. tuberculosis H37Rv in the presence of cholesterol as the sole carbon source under exponential growth and in two in vitro dormancy phases (NRP1 and NRP2). Using 2D-PAGE, we detected that M. tuberculosis expressed a high diversity of proteins in both exponential and non-replicative phases. We also found that cholesterol was involved in the overexpression of some proteins related to sulfur metabolism (CysA2), electron transport (FixB), cell wall synthesis (Ald), iron storage (BfrB), protein synthesis (Tig and EF-Tu) and dormancy maintenance (HspX and TB 31.7). According to our results we propose that proteins Ald, BfrB, FadA5 and TB31.7 are likely to play a fundamental role during in vitro dormancy of M. tuberculosis in the presence of cholesterol, helping to counteract its intracellular hostile microenvironment.

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