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

Hartkoorn, Ruben C.; Sala, Claudia; Magnet, Sophie; Chen, Jeffrey M.; Pojer, Florence; Cole, Stewart T.

doi:10.1128/jb.00687-10

Cited by 13 publications

(22 citation statements)

References 33 publications

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“…1B). As observed previously (15), the induction of sigF in both H37Rv::pMYsigF and H37Rv(⌬rsbW/ sigF)::pMYsigF brought about a decrease in growth rate after 24 h, whereas control strains were not affected.…”

Section: Resultssupporting

confidence: 61%

“…To identify the SigF promoters for the SigF targets validated above, 40-bp sequences upstream of the identified TSPs were in-spected and, in all cases, the consensus sequence, GGTTT-N (15)(16)(17) -GGGTA, was identified (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

“…P-IA was purified from pyostatin tablets as described previously (15). Rabbit anti-SigF polyclonal antibodies were kindly provided by Ida Rosenkrands (Statens Serum Institut, Copenhagen, Denmark), and a mouse anti-HbhA polyclonal antibody was provided by Giovanni Delogu (Catholic University of the Sacred Heart, Rome, Italy).…”

Section: Methodsmentioning

confidence: 99%

“…All experiments were performed in either M. tuberculosis strain H37Rv or its isogenic mutant, H37Rv(⌬rsbW/sigF), lacking the rsbW and sigF operon, as described previously (15). These strains were transformed with either pMY769, an empty pristinamycin IA (P-IA)-inducible integrating vector, or pMYsigF, a pMY769 derivative with a P-IA-inducible sigF gene (15) to create H37Rv::pMY769, H37Rv::pMYsigF, H37Rv(⌬rsbW/sigF)::pMY769, and H37Rv(⌬rsbW/sigF)::pMYsigF. The strains were routinely grown in Middlebrook 7H9 medium supplemented with 10% albumin-dextrosecatalase (ADC), 0.2% glycerol, and 0.05% Tween 80.…”

Section: Methodsmentioning

confidence: 99%

“…To analyze the total protein content, bacteria were harvested, and the total cell extract was subjected to SDS-PAGE for Simply Blue staining or Western blot analysis as described previously (15). Protein bands were excised from the gel and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis following trypsin or chymotrypsin digestion.…”

Section: Protein Gels and Western Blot Analysismentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Protein Gels and Western Blot Analysismentioning

confidence: 99%

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Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

et al. 2012

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Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

et al. 2015

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In Mycobacterium smegmatis, sigF is widely expressed during different growth stages and plays role in adaptation to stationary phase and oxidative stress. Using a sigF deletion mutant of M. smegmatis mc2155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoid pigmentation which rendered increased susceptibility to H2O2 induced oxidative stress in M. smegmatis. SigF modulates the cell surface architecture and lipid biosynthesis extending the repertoire of SigF function in this species. M. smegmatis SigF regulon included variety of genes expressed during exponential and stationary phases of growth and those responsible for oxidative stress, lipid biosynthesis, energy, and central intermediary metabolism. Furthermore, we report the identification of a SigF antagonist, an anti‐sigma factor (RsbW), which upon overexpression in M. smegmatis wild type strain produced a phenotype similar to M. smegmatis mc2155 ΔsigF strain. The SigF‐anti‐SigF interaction is duly validated using bacterial two‐hybrid and pull down assays. In addition, anti‐sigma factor antagonists, RsfA and RsfB were identified and their interactions with anti‐sigma factor were experimentally validated. Identification of these proteins will help decode regulatory circuit of this alternate sigma factor.

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Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation

Chauhan,

Baptista,

Arsh

et al. 2024

Biochemistry

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Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters' positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.

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

Cited by 13 publications

References 33 publications

Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation

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