scholarly journals Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae

2003 ◽  
Vol 185 (1) ◽  
pp. 349-358 ◽  
Author(s):  
Ping Luo ◽  
Donald A. Morrison
Keyword(s):  
Streptococcus Pneumoniae ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Consensus Sequence ◽  
Molar Ratio ◽  
Alternative Sigma Factor ◽  
Dna Uptake ◽  
Promoter Regions ◽  
Overexpression Strain ◽  
Peptide Pheromone

ABSTRACT Natural transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an Escherichia coli overexpression strain and core RNA polymerase was purified from a comX-deficient S. pneumoniae strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes ssbB, cinA, cglA, celA, and dalA and was inhibited by anti-ComX antibody, but not by anti-σ70 antibody. Western blotting using antibodies specific for ComX, σ70, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and σA remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.

scholarly journals Competence for Genetic Transformation in Streptococcus pneumoniae: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW

2009 ◽  
Vol 191 (10) ◽  
pp. 3359-3366 ◽  
Author(s):  
Andrew Piotrowski ◽  
Ping Luo ◽  
Donald A. Morrison
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Transformation ◽  
Sigma Factor ◽  
Transcriptional Activity ◽  
Physiological State ◽  
Alternative Sigma Factor ◽  
Rapid Loss ◽  
Gene Encoding ◽  
Peptide Pheromone

ABSTRACT Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other “early” genes. ComX, a transient alternative sigma factor, drives transcription of “late” genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.

scholarly journals Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis

2020 ◽  
Vol 48 (10) ◽  
pp. 5332-5348
Author(s):  
Taylor M Nye ◽  
Lieke A van Gijtenbeek ◽  
Amanda G Stevens ◽  
Jeremy W Schroeder ◽  
Justin R Randall ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bacillus Subtilis ◽  
Consensus Sequence ◽  
Dna Uptake ◽  
Cellular Functions ◽  
Promoter Regions ◽  
Gram Positive Bacteria ◽  
Genome Wide ◽  
Domains Of Life ◽  
Reporter Assays

Abstract The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

scholarly journals Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway inBacillus subtilis

2011 ◽  
Vol 77 (7) ◽  
pp. 2399-2405 ◽  
Author(s):  
Junfeng Xue ◽  
Birgitte K. Ahring
Keyword(s):  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Stress Factors ◽  
Alternative Sigma Factor ◽  
Wild Type ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Overexpression Strain ◽  
As Stress

ABSTRACTTo enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacteriumBacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed inB. subtilisDSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H2O2) stress, on isoprene production were further examined. Heat, salt, and H2O2induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

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

2007 ◽  
Vol 190 (2) ◽  
pp. 699-707 ◽  
Author(s):  
Jong-Hee Lee ◽  
Petros C. Karakousis ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Ribosomal Proteins ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription Analysis ◽  
Recombinant Strains ◽  
Regulatory Cascade

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.

scholarly journals DksA and ppGpp Regulate the σS Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Mary E. Girard ◽  
Saumya Gopalkrishnan ◽  
Elicia D. Grace ◽  
Jennifer A. Halliday ◽  
Richard L. Gourse ◽  
...  
Keyword(s):  
Stress Response ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Bacterial Species ◽  
Adaptor Protein ◽  
Large Family ◽  
Alternative Sigma Factor ◽  
Term Survival ◽  
Content Type

ABSTRACT σS is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σS general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σS levels transcriptionally, translationally, and posttranslationally in Escherichia coli. It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σS synthesis, thus playing an important role in activation of the σS stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σS levels. In addition, based on effects of DksAN88I, a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σS activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σS and also increase transcription from other promoters, including PdsrA and PiraP. By improving the translation and stabilization of σS, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σS. Understanding the factors governing σS activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σS levels.

scholarly journals The alternative sigma factor σX mediates competence shut-off at the cell pole in Streptococcus pneumoniae

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Calum HG Johnston ◽  
Anne-Lise Soulet ◽  
Matthieu Bergé ◽  
Marc Prudhomme ◽  
David De Lemos ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Sigma Factor ◽  
Feedback Loop ◽  
Genetic Circuit ◽  
Negative Feedback Loop ◽  
Alternative Sigma Factor ◽  
Bacterial Differentiation ◽  
Σ Factor ◽  
Spatiotemporal Localization

Competence is a widespread bacterial differentiation program driving antibiotic resistance and virulence in many pathogens. Here, we studied the spatiotemporal localization dynamics of the key regulators that master the two intertwined and transient transcription waves defining competence in Streptococcus pneumoniae. The first wave relies on the stress-inducible phosphorelay between ComD and ComE proteins, and the second on the alternative sigma factor σX, which directs the expression of the DprA protein that turns off competence through interaction with phosphorylated ComE. We found that ComD, σX and DprA stably co-localize at one pole in competent cells, with σX physically conveying DprA next to ComD. Through this polar DprA targeting function, σX mediates the timely shut-off of the pneumococcal competence cycle, preserving cell fitness. Altogether, this study unveils an unprecedented role for a transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit.

scholarly journals Crl, a Low Temperature-induced Protein inEscherichia coliThat Binds Directly to the Stationary Phase σ Subunit of RNA Polymerase

2004 ◽  
Vol 279 (19) ◽  
pp. 19540-19550 ◽  
Author(s):  
Alexandre Bougdour ◽  
Cécile Lelong ◽  
Johannes Geiselmann
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Low Temperature ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Physiological Signals ◽  
Alternative Sigma Factor ◽  
Transcription Rate ◽  
Stress Response Genes

The alternative sigma factor σS(RpoS) ofEscherichia coliRNA polymerase regulates the expression of stationary phase and stress-response genes. σSis also required for the transcription of the cryptic genescsgBAthat encode the subunits of the curli proteins. The expression of thecsgBAgenes is regulated in response to a multitude of physiological signals. In stationary phase, these genes are transcribed by the σSfactor, and expression of the operon is enhanced by the small protein Crl. It has been shown that Crl stimulates the activity of σS, leading to an increased transcription rate of a subset of genes of therpoSregulon in stationary phase. However, the underlying molecular mechanism has remained elusive. We show here that Crl interacts directly with σSand that this interaction promotes binding of the σSholoenzyme (EσS) to thecsgBApromoter. Expression of Crl is increased during the transition from growing to stationary phase. Crl accumulates in stationary phase cells at low temperature (30 °C) but not at 37 °C. We therefore propose that Crl is a second thermosensor, besides DsrA, controlling σSactivity.

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