Roles of SigB and SigF in the Mycobacterium tuberculosis Sigma Factor Network

ABSTRACTTo characterize the roles of SigB and SigF in sigma factor regulation inMycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpresssigBandsigF.Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes aftersigBinduction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression ofsigBandsigF. Overexpression ofsigBresulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction ofsigBdid not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of theM. tuberculosissigma factor regulatory cascade. Analysis of the 5′-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of bothsigB(Rv2710) and the gene following it,ideR(Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression ofsigFrevealed that only thesigCgene was significantly upregulated 6 and 12 h aftersigFinduction. The previously identified SigF promoter consensus sequence AGTTTG-N15-GGGTTT was identified in the 5′ UTR of thesigCgene, and SigF-dependent in vitro transcription of the promoter upstream ofsigCwas confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression ofsigBandsigFresulted in reduced rates ofM. tuberculosisintracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00687-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5472-5479 ◽

Cited By ~ 8

Author(s):

Ruben C. Hartkoorn ◽

Claudia Sala ◽

Sophie J. Magnet ◽

Jeffrey M. Chen ◽

Florence Pojer ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Stationary Phase ◽

Sigma Factor ◽

Potent Inhibitor ◽

Sigma Factors ◽

Antituberculosis Drugs ◽

Axenic Cultures

ABSTRACT 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.

Download Full-text

Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819682116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8310-8319 ◽

Cited By ~ 52

Author(s):

Patricia Sanchez-Vazquez ◽

Colin N. Dewey ◽

Nicole Kitten ◽

Wilma Ross ◽

Richard L. Gourse

Keyword(s):

Rna Polymerase ◽

Dna Sequences ◽

Binding Sites ◽

Promoter Sequence ◽

In Vitro Transcription ◽

Transcriptional Responses ◽

Transcription Analysis ◽

Genome Wide

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

Download Full-text

fleQ, the Gene Encoding the Major Flagellar Regulator of Pseudomonas aeruginosa, Is σ70 Dependent and Is Downregulated by Vfr, a Homolog of Escherichia coli Cyclic AMP Receptor Protein

Journal of Bacteriology ◽

10.1128/jb.184.19.5240-5250.2002 ◽

2002 ◽

Vol 184 (19) ◽

pp. 5240-5250 ◽

Cited By ~ 74

Author(s):

Nandini Dasgupta ◽

Evan P. Ferrell ◽

Kristen J. Kanack ◽

Susan E. H. West ◽

Reuben Ramphal

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Binding Site ◽

Caulobacter Crescentus ◽

Bacterial Species ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Receptor Protein ◽

Regulatory Cascade

ABSTRACT The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a σ70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5′-AATTGACTAATCGTTCACATTTG-3′. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative −10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli σ70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this −10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the −10 region to initiate transcription.

Download Full-text

Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2543-2550.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2543-2550

Author(s):

D F Bogenhagen ◽

B K Yoza

Keyword(s):

Mitochondrial Dna ◽

Xenopus Laevis ◽

Rna Polymerase ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Xenopus Laevis Oocytes ◽

Mitochondrial Rna ◽

Bidirectional Promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

Download Full-text

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing σ A , the major sigma factor in growing cells

Molecular Microbiology ◽

10.1046/j.1365-2958.1997.4751843.x ◽

1997 ◽

Vol 25 (3) ◽

pp. 463-472 ◽

Cited By ~ 13

Author(s):

Dvora Biran ◽

Lee Kroos

Keyword(s):

Rna Polymerase ◽

Sigma Factor ◽

Myxococcus Xanthus ◽

In Vitro Transcription

Download Full-text

Transcriptional analysis of thePseudomonas aeruginosa toxAregulatory geneptxR

Canadian Journal of Microbiology ◽

10.1139/w05-138 ◽

2006 ◽

Vol 52 (4) ◽

pp. 343-356 ◽

Cited By ~ 7

Author(s):

Jane A Colmer-Hamood ◽

Hironori Aramaki ◽

Jennifer M Gaines ◽

Abdul N Hamood

Keyword(s):

Pseudomonas Aeruginosa ◽

Rna Polymerase ◽

In Vitro Transcription ◽

Transcriptional Analysis ◽

Upstream Region ◽

Transcription Analysis ◽

Iron Deficient ◽

P2 Promoter ◽

Microaerobic Conditions

The expression of the exotoxin A gene (toxA) in Pseudomonas aeruginosa is a complicated process that involves several regulators, including ptxR, which enhances toxA expression by 4- to 5-fold. Available evidence suggests that ptxR is expressed from two separate promoters, P1 and P2. Previous evidence indicated the presence, within the ptxR upstream region, of binding sites for several regulatory proteins, including PtxS, which negatively regulates ptxR expression. We utilized nested deletion and in vitro transcription analyses to examine the regulation of ptxR expression. The results from nested deletion analysis suggest that under aerobic conditions in iron-deficient medium, ptxR expression follows a biphasic curve that involves the P1 promoter only. Iron eliminated the second peak of ptxR expression but did not affect expression from the P2 promoter. Under microaerobic conditions, iron represses ptxR expression from subclones that carry P1 alone or P2 alone at both early and late stages of growth. Under anaerobic conditions, ptxR expression increases considerably. In addition, our results suggest that different segments of the ptxR upstream region play specific roles in ptxR expression; their deletion caused variations in the level as well as the pattern of ptxR expression. Our results also indicate that negative regulation of ptxR expression by PtxS does not occur through the PtxS binding site within the ptxR–ptxS intergenic region. In vitro transcription analysis using σ70-reconstituted P. aeruginosa RNA polymerase produced one transcript that closely resembles T1, indicating that P1 is recognized by σ70. RNA polymerase reconstituted with either RpoS or AlgU produced no transcripts. However, a transcript was produced by RpoH-reconstituted RNA polymerase.Key words: ptxR, regulation, Pseudomonas aeruginosa, PAO1.

Download Full-text