Evolution of the Mycobacterial SigK Regulon

Abstract

Previous studies have established that members of the Mycobacterium tuberculosis complex exhibit variable production of the antigenic proteins MPT70 and MPT83 due to mutations in their positive regulator, SigK (sigma factor K), and their negative regulator, RskA (regulator of sigma K). To further understand this highly specific SigK-controlled regulon, we have undertaken evolutionary studies to determine the presence of homologues of SigK-regulated genes in other organisms and to predict its transcriptional network. Evolutionary analysis indicates that the positive and negative regulators are conserved across many organisms, but that the genes under their control are variable. Moreover, the addition, loss, and movement of various genes in the mpt70/83 locus suggest that these genes are unlikely to be cotranscribed. To test predictions from sequence analysis, we have used promoter luciferase fusions and Northern blots to show that the majority of genes in this locus have their own promoters, of which a subset are SigK regulated (mpt83, dipZ, mpt70, and Rv0449c). Next, we have shown that the intracellular inducibility of mpt70 and mpt83 is a conserved property, shared between M. tuberculosis and Mycobacterium marinum. In addition, we have shown that SigK and RskA from an environmental mycobacterium isolate (M. gilvum PYR-GCK) complemented the regulatory activity of M. tuberculosis ⌬sigK rskA. Together, our data indicate that the regulatory system SigK/RskA is conserved across the Mycobacterium genus, whereas the regulon under its control varies considerably across species.Mycobacterium tuberculosis is an extremely successful pathogen of humans, infecting an estimated one-third of the world's population. This capacity to persist in the host, despite the dynamic alterations effected by the host immune response, testifies to an evolved capacity of the organism to regulate its genetic expression to the conditions encountered during infection. Indeed, a number of studies have demonstrated that disruption of M. tuberculosis regulatory elements, such as sigma factors, results in reduced virulence in experimental models (21,28,30).The determination of genomic sequences for a number of nonpathogenic mycobacteria has revealed that homologues of many genes, including regulatory elements, are often present in organisms that are not virulent to humans. For instance, comparative analysis has demonstrated conservation of biochemical pathways and a secretory system important for full virulence of M. tuberculosis (7,10). This indicates that genetic elements and the proteins they encode that exist in other mycobacteria either serve an important function in M. tuberculosis pathogenicity or have evolved a specific role in M. tuberculosis complex organisms. As a number of predicted regulatory elements are also conserved between environmental mycobacteria and M. tuberculosis, it follows that an evolutionary analysis may guide predictions about the origins of such elements, the transcriptional networks they control, and the stimuli that...

The sigma factors of Mycobacterium tuberculosis: regulation of the regulators

“…In slow‐growing pathogenic mycobacteria, an additional gene, dipZ , is inserted between the two mpt83 paralogs. The σ K regulon is atypically small; however, the number and identity of σ K ‐regulated genes varies across different mycobacterial species [101]. The role and trigger of this regulon in M. tuberculosis pathogenicity merits investigation.…”

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

“…In turn, this correlates with a dramatic shift in the overall pattern of DosR regulon gene expression within these strains that, bearing in mind the high energetic cost likely associated with maintaining this phenotype, almost certainly affords some form of survival or fitness advantage within the environment of the host. Other host-adaptive regulatory mutations described within the wider M. tuberculosis complex, such as those in the PhoPR and SigK systems, have been associated with broad evolutionary shifts linked to specific host species (73,74). It will be intriguing in the future to determine if the lineage-specific DosR trait detailed herein also represents a bacterial adaptation to a specific host population or hostassociated selective pressure.…”

Region of Difference 2 Contributes to Virulence ofMycobacterium tuberculosis

“…For H37Rv:⌬RD2::p8586, we determined the expression of both Rv1985c and Rv1986 in the wild-type and complemented strains using quantitative reverse transcription-PCR (qRT-PCR) by following procedures described elsewhere (39). Briefly, RNA was extracted from cultures in log-phase growth (optical density at 600 nm [OD 600 ] of 0.4 to 0.6) using a modified phenol-chloroform extraction method, which has been described previously (6), and levels of Rv1985c and Rv1986 were normalized to the amount of sigA RNA (www.molepi .mcgill.ca).…”

“…Primers used in this study are listed in Table 1. Plasmids were electroporated into M. tuberculosis H37Rv:⌬RD2, kanamycin-resistant clones were selected, and the presence of the plasmid was assessed by PCR as has been previously described (39).…”