Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

Abstract

bIn Mycobacterium tuberculosis the alternative sigma factor SigF controls the expression of a particular subset of genes by altering RNA polymerase specificity. Here, we utilize two genome-wide approaches to identify SigF-binding sites: chromatin immunoprecipitation (ChIP-on-chip) and microarray analysis of SigF-mediated transcripts. Since SigF is not an abundant protein in the logarithmic phase of growth, a pristinamyin IA-inducible system was used to control its expression. We identified 67 highaffinity SigF-binding sites and 16 loci where a SigF promoter directs the expression of a transcript. These loci include sigF itself, genes involved in lipid and intermediary metabolism and virulence, and at least one transcriptional regulator (Rv2884), possibly acting downstream of SigF. In addition, SigF was also found to direct the transcription of the gene for small RNA F6. Many loci were also found where SigF may be involved in antisense transcription, and in two cases (Rv1358 and Rv1870c) the SigF-dependent promoter was located within the predicted coding sequence. Quantitative PCR confirmed the microarray findings and 5=-rapid amplification of cDNA ends was used to map the SigF-specific transcriptional start points. A canonical SigF consensus promoter sequence GGTTT-N (15-17) -GGGTA was found prior to 11 genes. Together, these data help to define the SigF regulon and show that SigF not only governs expression of proteins such as the virulence factor, HbhA, but also impacts novel functions, such as noncoding RNAs and antisense transcripts.

A Walk into the LuxR Regulators of Actinobacteria: Phylogenomic Distribution and Functional Diversity

“…Another of these proteins, encoded by Rv0386, was shown to have an alternative substrate-binding mechanism regarding its cyclase activity [41], and infection with M. tuberculosis that do not express Rv0386 resulted in a decreased bacterial-derived intramacrophage cAMP, tumour necrosis factor- (TNF) production and bacterial survival [42]. Finally, a M. tuberculosis strain overexpressing an alternative sigma factor, SigF, whose absence causes a partially attenuated phenotype, was found to have a 14-fold increase in the anti-sense mRNA transcript of Rv1358, another CHD+LuxR encoding gene [43].These studies further support the association between these regulators and virulence in mycobacteria, while raising the question of the role played by the LuxR domain in these proteins and suggesting cAMP or cGMP as potential signals. It should be stressed that, as in almost all Actinobacteria, the knowledge on QS in Mycobacterium genus is scarce and limited to indirect evidences, such as the induction of biofilm formation in M. avium after exposure to AI-2 [44] and the QS-like expression of the tissue-damage related transcriptional regulator WhiB3 in M. tuberculosis [45].…”

Global Analysis of the Regulon of the Transcriptional Repressor LexA, a Key Component of SOS Response in Mycobacterium tuberculosis

“…Also, 60% of the identified binding sites for the alternative σ factor, SigF, of M. tuberculosis were not linked with effects on transcription (41). Here, five LexA binding peaks were identified with genes that are not DNA damage-inducible or the induction remained in a RecA mutant strain, in which the LexA repression should not be able to be lifted.…”

Non-coding RNA and its potential role in Mycobacterium tuberculosis pathogenesis

“…MTS194 is encoded in the intergenic region between two genes involved in lipid degradation, Rv0243 and Rv0244c. It is transcribed by the starvation associated alternative sigma factor, SigF and the MTS194 promoter has recently been shown to represent the highest occupancy of this sigma factor suggesting that expression of this sRNA has high priority under certain conditions such as starvation 76 . Overexpression of MTS194 leads to reduced growth in M. tuberculosis , but although an MTS194 homolog is present in the distantly related, non-pathogenic Mycobaterium smegmatis , overexpression of MTS194 in M. smegmatis has no obvious phenotype 29 …”