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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m314145200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19540-19550 ◽

Cited By ~ 91

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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The Alternative Sigma Factor SigB Is Required for the Pathogenicity of Bacillus thuringiensis

Journal of Bacteriology ◽

10.1128/jb.00265-20 ◽

2020 ◽

Vol 202 (21) ◽

Author(s):

Stéphanie Henry ◽

Didier Lereclus ◽

Leyla Slamti

Keyword(s):

Life Cycle ◽

Stress Response ◽

Bacillus Thuringiensis ◽

Sigma Factor ◽

Oral Route ◽

Alternative Sigma Factor ◽

Gram Positive ◽

Content Type ◽

General Stress Response

ABSTRACT To adapt to changing and potentially hostile environments, bacteria can activate the transcription of genes under the control of alternative sigma factors, such as SigB, a master regulator of the general stress response in several Gram-positive species. Bacillus thuringiensis is a Gram-positive spore-forming invertebrate pathogen whose life cycle includes a variety of environments, including plants and the insect hemocoel or gut. Here, we assessed the role of SigB during the infectious cycle of B. thuringiensis in a Galleria mellonella insect model. We used a fluorescent reporter coupled to flow cytometry and showed that SigB was activated in vivo. We also showed that the pathogenicity of the ΔsigB mutant was severely affected when inoculated via the oral route, suggesting that SigB is critical for B. thuringiensis adaptation to the gut environment of the insect. We could not detect an effect of the sigB deletion on the survival of the bacteria or on their sporulation efficiency in the cadavers. However, the gene encoding the pleiotropic regulator Spo0A was upregulated in the ΔsigB mutant cells during the infectious process. IMPORTANCE Pathogenic bacteria often need to transition between different ecosystems, and their ability to cope with such variations is critical for their survival. Several Gram-positive species have developed an adaptive response mediated by the general stress response alternative sigma factor SigB. In order to understand the ecophysiological role of this regulator in Bacillus thuringiensis, an entomopathogenic bacterium widely used as a biopesticide, we sought to examine the fate of a ΔsigB mutant during its life cycle in the natural setting of an insect larva. This allowed us, in particular, to show that SigB was activated during infection and that it was required for the pathogenicity of B. thuringiensis via the oral route of infection.

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A Mutant RNA Polymerase Activates the General Stress Response, Enabling Escherichia coli Adaptation to Late Prolonged Stationary Phase

mSphere ◽

10.1128/msphere.00092-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Pabitra Nandy ◽

Savita Chib ◽

Aswin Seshasayee

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Stationary Phase ◽

Sigma Factor ◽

Slow Growth ◽

Content Type ◽

General Stress Response ◽

E Coli ◽

General Stress ◽

Small Colony

ABSTRACT Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σS. The RpoC(A494V) mutation upregulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase. IMPORTANCE An important general mechanism of a bacterium’s adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

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A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation

Journal of Bacteriology ◽

10.1128/jb.00212-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 3

Author(s):

David M. Rothstein ◽

David Lazinski ◽

Marcia S. Osburne ◽

Abraham L. Sonenshein

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Sigma Factor ◽

Rna Synthesis ◽

Bacterial Species ◽

Ethanol Stress ◽

Temperature Sensitive ◽

Content Type ◽

Promoter Recognition ◽

Bacillis Subtilis

ABSTRACT Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.

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Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

mSphere ◽

10.1128/msphere.00728-20 ◽

2020 ◽

Vol 5 (5) ◽

Cited By ~ 1

Author(s):

Ilse M. Boekhoud ◽

Annika-Marisa Michel ◽

Jeroen Corver ◽

Dieter Jahn ◽

Wiep Klaas Smits

Keyword(s):

Hydrogen Peroxide ◽

Stress Response ◽

Sigma Factor ◽

Gene Activation ◽

Luciferase Reporter ◽

Alternative Sigma Factor ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

Sigma B

ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation. IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

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Long-Term Survival of Borrelia burgdorferi Lacking the Hibernation Promotion Factor Homolog in the Unfed Tick Vector

Infection and Immunity ◽

10.1128/iai.00925-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4800-4810 ◽

Cited By ~ 7

Author(s):

Lisa Fazzino ◽

Kit Tilly ◽

Daniel P. Dulebohn ◽

Patricia A. Rosa

Keyword(s):

Borrelia Burgdorferi ◽

Stationary Phase ◽

Bacterial Species ◽

Protein Secondary Structure ◽

Mammalian Host ◽

Term Survival ◽

Content Type ◽

Protein Levels ◽

And Function

Borrelia burgdorferi, a causative agent of Lyme borreliosis, is a zoonotic pathogen that survives in nutrient-limited environments within a tick, prior to transmission to its mammalian host. Survival under these prolonged nutrient-limited conditions is thought to be similar to survival during stationary phase, which is characterized by growth cessation and decreased protein production. Multiple ribosome-associated proteins are implicated in stationary-phase survival ofEscherichia coli. These proteins include hibernation-promoting factor (HPF), which dimerizes ribosomes and prevents translation of mRNA. Bioinformatic analyses indicate thatB. burgdorferiharbors anhpfhomolog, thebb0449gene. BB0449 protein secondary structure modeling also predicted HPF-like structure and function. However, BB0449 protein was not localized in the ribosome-associated protein fraction ofin vitro-grownB. burgdorferi. In wild-typeB. burgdorferi,bb0449transcript and BB0449 protein levels are low during various growth phases. These results are inconsistent with patterns of synthesis of HPF-like proteins in other bacterial species. In addition, two independently derivedbb0449mutants successfully completed the mouse-tick infectious cycle, indicating thatbb0449is not required for prolonged survival in the nutrient-limited environment in the unfed tick or any other stage of infection byB. burgdorferi. We suggest either that BB0449 is associated with ribosomes under specific conditions not yet identified or that BB0449 ofB. burgdorferihas a function other than ribosome conformation modulation.

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

Journal of Bacteriology ◽

10.1128/jb.00277-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 1

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.

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A mutant RNA polymerase activates the general stress response, enabling E. coli adaptation to late prolonged stationary phase

10.1101/2020.01.27.922484 ◽

2020 ◽

Author(s):

Pabitra Nandy ◽

Savita Chib ◽

Aswin Seshasayee

Keyword(s):

Stress Response ◽

Rna Polymerase ◽

Stationary Phase ◽

Sigma Factor ◽

Slow Growth ◽

General Mechanism ◽

General Stress Response ◽

E Coli ◽

General Stress ◽

Small Colony

AbstractEscherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after three weeks o of maintaining an E. coli K12 population in stationary phase. This variant displayed a small colony phenotype, slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC (A494V) mutation confers the slow growth and small colony phenotype to this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary phase sigma factor σS. The RpoC (A494V) mutation up-regulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly, or via mutations such as RpoC (A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase.ImportanceAn important general mechanism of bacterial adaptation to its environment involves adjusting the balance between growing fast, and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favour of the general stress response about two weeks later. This can be established by direct mutations in the master regulator of the general stress response, or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance though the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

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Role of the Alternative Sigma Factor ςS in Expression of the AlkS Regulator of thePseudomonas oleovorans Alkane Degradation Pathway

Journal of Bacteriology ◽

10.1128/jb.181.6.1748-1754.1999 ◽

1999 ◽

Vol 181 (6) ◽

pp. 1748-1754 ◽

Cited By ~ 32

Author(s):

Inés Canosa ◽

Luis Yuste ◽

Fernando Rojo

Keyword(s):

Rna Polymerase ◽

Stationary Phase ◽

Sigma Factor ◽

Degradation Pathway ◽

Exponential Phase ◽

Alternative Sigma Factor ◽

Alkane Degradation ◽

Gene Coding ◽

Low Levels ◽

High Level

ABSTRACT The AlkS protein activates transcription from the PalkBpromoter, allowing the expression of a number of genes required for the assimilation of alkanes in Pseudomonas oleovorans. We have identified the promoter from which the alkS gene is transcribed, PalkS, and analyzed its expression under different conditions and genetic backgrounds. Transcription fromPalkS was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. ThePalkS −10 region was similar to the consensus described for promoters recognized by Escherichia coli RNA polymerase bound to the alternative sigma factor ςS, which directs the expression of many stationary-phase genes. Reporter strains containing PalkS-lacZ transcriptional fusions showed thatPalkS promoter is very weakly expressed in aPseudomonas putida strain bearing an inactivated allele of the gene coding for ςS, rpoS. WhenPalkS was transferred to E. coli, transcription started at the same site and expression was higher in stationary phase only if ςS-RNA polymerase was present. The low levels of AlkS protein generated in the absence of ςS were enough to support a partial induction of the PalkB promoter. The −10 and −35 regions of PalkS promoter also show some similarity to the consensus recognized by ςD-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase PalkS is probably recognized both by ςD-RNA polymerase (inefficiently) and by ςS-RNA polymerase (present at low levels), leading to low-level expression of the alkS gene. ςS-RNA polymerase would be responsible for the high level of activity ofPalkS observed in stationary phase.

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Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502

Applied and Environmental Microbiology ◽

10.1128/aem.04036-12 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3867-3869 ◽

Cited By ~ 12

Author(s):

Elias Dahlsten ◽

David Kirk ◽

Miia Lindström ◽

Hannu Korkeala

Keyword(s):

Stress Tolerance ◽

Sigma Factor ◽

Clostridium Botulinum ◽

Alternative Sigma Factor ◽

Strain Atcc ◽

Content Type ◽

Osmotic Stress Tolerance ◽

Group I ◽

Temperature Downshift

ABSTRACTThe role of the alternative sigma factor SigK in cold and osmotic stress tolerance ofClostridium botulinumATCC 3502 was demonstrated by induction ofsigKafter temperature downshift and exposure to hyperosmotic conditions and by impaired growth of thesigKmutants under the respective conditions.

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