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

2020 ◽  
Author(s):  
Calum Johnston ◽  
Anne-Lise Soulet ◽  
Matthieu Berge ◽  
Marc Prudhomme ◽  
David De Lemos ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sigma Factor ◽  
Protein Targeting ◽  
Genetic Circuit ◽  
Alternative Sigma Factor ◽  
Cell Periphery ◽  
Cell Pole ◽  
Bacterial Transcription ◽  
Σ Factor ◽  
Species Specific

SummaryBacterial competence for genetic transformation is a well-known species-specific differentiation program driving genome plasticity, antibiotic resistance and virulence in many pathogens. How competence regulation is spatiotemporally integrated in the cell is ill-defined. Here, we unraveled the localization dynamics of the key regulators that master the two intertwined transcription waves controlling competence in Streptococcus pneumoniae. The first wave relies on a stress-inducible phosphorelay system, made up of the ComD and ComE proteins, and the second is directed by an alternative sigma factor, σX, which includes in its regulon the DprA protein that turns off competence through interaction with phosphorylated ComE. Remarkably, we found that ComD, σX and DprA stably co-localize at a single cell pole over the competence period. In contrast, ComE assembles into dynamic patches in the cell periphery, colocalizing temporarily with DprA and ComD at the pole. Furthermore, we provide evidence that σX directly conveys DprA polar anchoring. Through this protein targeting function, σX is shown to be actively involved in the timely shut-off of the competence cycle, hence preserving cell fitness. Altogether, this study unveils an unprecedented role for a bacterial transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit.

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 Author response: The alternative sigma factor σX mediates competence shut-off at the cell pole in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Calum HG Johnston ◽  
Anne-Lise Soulet ◽  
Matthieu Bergé ◽  
Marc Prudhomme ◽  
David De Lemos ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sigma Factor ◽  
Author Response ◽  
Alternative Sigma Factor ◽  
Cell Pole

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 Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σABypass the ComW Requirement for Late Gene Expression

2016 ◽  
Vol 198 (17) ◽  
pp. 2370-2378 ◽  
Author(s):  
Yanina Tovpeko ◽  
Junqin Bai ◽  
Donald A. Morrison
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Genetic Transformation ◽  
Sigma Factor ◽  
Specific Protein ◽  
Late Gene ◽  
Late Gene Expression ◽  
Content Type ◽  
Σ Factor

ABSTRACTStreptococcus pneumoniaeis able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the onlyS. pneumoniaealternative σ factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW. The ΔcomWmutant is 104-fold deficient in the yield of recombinants, 10-fold deficient in the amount of σXactivity, and 10-fold deficient in the amount of σXprotein. The critical role of ComW during transformation can be partially obviated by σAmutations clustered on surfaces controlling affinity for core RNA polymerase (RNAP). While strains harboring σAmutations in thecomWmutant background were transforming at higher rates, the mechanism of transformation restoration was not clear. To investigate the mechanism of transformation restoration, we measured late gene expression in σA* suppressor strains. Restoration of late gene expression was observed in ΔcomWσA* mutants, indicating that a consequence of the σA* mutations is, at least, to restore σXactivity. Competence kinetics were normal in ΔcomWσA* strains, indicating that strains with restored competence exhibit the same pattern of transience as wild-type (WT) strains. We also identified a direct interaction between ComW and σXusing the yeast two-hybrid (Y2H) assay. Taken together, these data are consistent with the idea that ComW increases σXaccess to core RNAP, pointing to a direct role of ComW in σ factor exchange during genetic transformation. However, the lack of late gene shutoff in ΔcomWmutants also points to a potential new role for ComW in competence shutoff.IMPORTANCEThe sole alternative sigma factor of the streptococci, SigX, regulates development of competence for genetic transformation, a widespread mechanism of adaptation by horizontal gene transfer in this genus. The transient appearance of this sigma factor is strictly controlled at the levels of transcription and stability. This report shows that it is also controlled at the point of its substitution for SigA by a second transient competence-specific protein, ComW.

scholarly journals An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Anna F. Wang Erickson ◽  
Padraig Deighan ◽  
Cinthia P. Garcia ◽  
Robert O. J. Weinzierl ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Sigma Factor ◽  
Coiled Coil ◽  
Sigma Factors ◽  
Alternative Sigma Factor ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Σ Factor

ABSTRACT Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β′ subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σH, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σH-directed gene expression but not in the activity of the stress-response sigma factor σB. Pulldown experiments showed that the mutant RNAP was defective in associating with σH but could still associate with σA and σB. The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β′ coiled coil that is specifically detrimental for interaction with σH. This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor. IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β′ subunit whereby RNAPL257P associates with some σ factors (σA and σB) and enables vegetative cell growth but is defective in utilization of σH and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.

scholarly journals Global transcriptional response to vancomycin in Mycobacterium tuberculosis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1093-1102 ◽  
Author(s):  
Roberta Provvedi ◽  
Francesca Boldrin ◽  
Francesco Falciani ◽  
Giorgio Palù ◽  
Riccardo Manganelli
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Surface Stress ◽  
Sigma Factor ◽  
Clustering Analysis ◽  
Transcriptional Response ◽  
Alternative Sigma Factor ◽  
Hierarchical Clustering Analysis ◽  
Physiological Mechanisms ◽  
Basal Resistance ◽  
Antibacterial Drugs

In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin–antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor σ E confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.

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