A recombinant<i>Mycobacterium tuberculosis in vitro</i>transcription system

Jacques, Jean-FranÃ§ois; Rodrigue, SÃ©bastien; Brzeziński, Ryszard; Gaudreau, Luc

doi:10.1111/j.1574-6968.2005.00071.x

Cited by 38 publications

(35 citation statements)

References 26 publications

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“…Thus, it is tempting to speculate that the two proteins may use the same promoter at some of these genes. The latter hypothesis is supported by the high level of homology between A and B and by the fact that the corresponding holoenzymes allowed transcription initiation at the same nucleotide in vitro (19). In spite of the unexpectedly low number of factor binding sites, our approach defined new target genes for many M. tuberculosis factors.…”

Section: Discussionmentioning

confidence: 62%

“…The F -dependent usfXP1 promoter is one of the few promoters that have been characterized both in vivo and in vitro (2,19). Moreover, F is negatively regulated by the anti-factor UsfX (2).…”

Section: Resultsmentioning

confidence: 99%

“…In vitro transcription assays were performed as described elsewhere (19), except that E. coli core RNA polymerase (RNAP) (Epicenter Biotechnologies) was used in combination with M. tuberculosis factors.…”

Section: Methodsmentioning

confidence: 99%

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Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

et al. 2007

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Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for F , C , and K , and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of factor genes. Our results revealed that many factors are expressed from autoregulated promoters.Several pathogenic mycobacteria cause infections that lead to tuberculosis or related diseases in a variety of organisms. For example, Mycobacterium tuberculosis infects nearly onethird of the human population (10), while Mycobacterium bovis infections in cattle are responsible for important agricultural losses (12). Advanced knowledge of the biology of these bacteria could significantly contribute to prevention and treatment of the associated diseases. A key step toward this end occurred with the publication of the complete M. tuberculosis and M. bovis genome sequences (8, 12). Several genes have also been interrupted in M. tuberculosis, leading to different outcomes for virulence (for reviews, see references 7 and 38). More recently, in many studies workers have reported gene expression profiles for various growth conditions and during different stages of infection in mice and human macrophages (21, 37, 41), providing new insights into the strategies used by the pathogens to adapt to various environments. Nevertheless, much remains to be discovered about the molecular mechanisms controlling the genetic programs in pathogenic mycobacteria.Thirteen factor genes were predicted based on the genome sequence of M. tuberculosis, and almost identical M. bovis counterparts were also predicted (25,35). Some of these genes may orchestrate critical responses in the pathogenesis of tuberculosis and therefore may be attractive targets for development of new drugs and vaccines. Indeed, in mouse models infections with sigC, sigD, sigE, sigF, sigh, and sigL mutant strains were all attenuated compared to infections with the wild-type strains (5,9,13,16,20,23,24,26,27,32,33,40). Still, the conditions leading to the activities of most factors remain elusive (23,35). Differential gene expression profiles were nonetheless obtained for the factor mutant strains, and consensus promoter sequences have been proposed (5,9,13,16,20,26,27,32,33,40). However, direct and indirect effec...

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Section: Discussionmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

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Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

et al. 2007

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“…Alternative strategies used to discover SigF-controlled genes were ChIP-on-chip (chromatin immunoprecipitation and hybridization to microarrays) analysis of SigF-binding sites in BCG (30) and application of a two-plasmid system in Escherichia coli to identify genes controlled by SigF (16). Overall, there is little agreement between the findings of these studies except for confirmation of the initial finding that SigF controls its own transcription (18).…”

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confidence: 43%

Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

et al. 2012

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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.

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“…Recombinant M. tuberculosis sigma factors are usually generated by refolding sigma factors from denatured inclusion bodies following protein induction in E. coli (13,14). Here we found it possible to generate sufficient amounts of recombinant SigA (rSigA) and SigF (rSigF) by inducing at a lower IPTG concentration and at a lower temperature, albeit requiring a larger culture volume (6 liters).…”

Section: Resultsmentioning

confidence: 84%

Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

et al. 2010

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The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

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A recombinantMycobacterium tuberculosis in vitrotranscription system

Cited by 38 publications

References 26 publications

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

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