scholarly journals Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

2020 ◽  
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 ◽  
Transcriptional Level ◽  
Alternative Sigma Factor ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Sigma B

AbstractIn 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 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 allowed for the refinement of the previously proposed σB regulon. At least 32% 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 run-off transcription of specific target genes (cd0350, cd3614, cd3605, 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.ImportanceSigma 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.

scholarly journals 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 ◽  
2020 ◽  
Vol 5 (5) ◽  
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.

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

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.

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 CtsR Is the Master Regulator of Stress Response Gene Expression in Oenococcus oeni

2005 ◽  
Vol 187 (16) ◽  
pp. 5614-5623 ◽  
Author(s):  
Cosette Grandvalet ◽  
Françoise Coucheney ◽  
Charlotte Beltramo ◽  
Jean Guzzo
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Molecular Chaperone ◽  
Oenococcus Oeni ◽  
Transcriptional Level ◽  
Class Iii ◽  
Heat Shock Genes ◽  
Gram Positive ◽  
Stress Genes ◽  
Gram Positive Bacteria

ABSTRACT Although many stress response genes have been characterized in Oenococcus oeni, little is known about the regulation of stress response in this malolactic bacterium. The expression of eubacterial stress genes is controlled both positively and negatively at the transcriptional level. Overall, negative regulation of heat shock genes appears to be more widespread among gram-positive bacteria. We recently identified an ortholog of the ctsR gene in O. oeni. In Bacillus subtilis, CtsR negatively regulates expression of the clp genes, which belong to the class III family of heat shock genes. The ctsR gene of O. oeni is cotranscribed with the downstream clpC gene. Sequence analysis of the O. oeni IOB 8413 (ATCC BAA-1163) genome revealed the presence of potential CtsR operator sites upstream from most of the major molecular chaperone genes, including the clp genes and the groES and dnaK operons. Using B. subtilis as a heterologous host, CtsR-dependent regulation of O. oeni molecular chaperone genes was demonstrated with transcriptional fusions. No alternative sigma factors appear to be encoded by the O. oeni IOB 8413 (ATCC BAA-1163) genome. Moreover, apart from CtsR, no known genes encoding regulators of stress response, such as HrcA, could be identified in this genome. Unlike the multiple regulatory mechanisms of stress response described in many closely related gram-positive bacteria, this is the first example where dnaK and groESL are controlled by CtsR but not by HrcA.

scholarly journals Identification of the σB Regulon of Bacillus cereus and Conservation of σB-Regulated Genes in Low-GC-Content Gram-Positive Bacteria

2007 ◽  
Vol 189 (12) ◽  
pp. 4384-4390 ◽  
Author(s):  
Willem van Schaik ◽  
Menno van der Voort ◽  
Douwe Molenaar ◽  
Roy Moezelaar ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Heat Shock ◽  
Bacillus Cereus ◽  
Sigma Factor ◽  
Phylogenetic Analyses ◽  
Gc Content ◽  
Alternative Sigma Factor ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Food Borne ◽  
Lower Expression

ABSTRACT The alternative sigma factor σB has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of σB-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being σB dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor σB and a crucial positive regulator of σB activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified σB-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the σB regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where σB of the B. cereus group was placed close to the ancestral form of σB in gram-positive bacteria. The data described in this study and previous studies in which the complete σB regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of σB-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their σB dependency in all four bacteria, suggesting that the σB regulon of the different gram-positive bacteria has evolved to perform niche-specific functions.

scholarly journals Sigma B Contributes to PrfA-Mediated Virulence in Listeria monocytogenes

2002 ◽  
Vol 70 (7) ◽  
pp. 3948-3952 ◽  
Author(s):  
Celine A. Nadon ◽  
Barbara M. Bowen ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Sigma Factor ◽  
Deletion Mutation ◽  
Alternative Sigma Factor ◽  
Regulatory Factor ◽  
Virulence Phenotype ◽  
Sigma B

ABSTRACT Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp 1 and prfAp 2) or from the upstream plcA promoter. We demonstrate that prfAp 2 is a functional σB-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor σB contributes to virulence in L. monocytogenes.

scholarly journals A Type IV-Secretion-Like System Is Required for Conjugative DNA Transport of Broad-Host-Range Plasmid pIP501 in Gram-Positive Bacteria

2007 ◽  
Vol 189 (6) ◽  
pp. 2487-2496 ◽  
Author(s):  
Mohammad Y. Abajy ◽  
Jolanta Kopeć ◽  
Katarzyna Schiwon ◽  
Michal Burzynski ◽  
Mike Döring ◽  
...  
Keyword(s):  
Host Range ◽  
Sequence Similarity ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Computer Assisted ◽  
Transcriptional Level ◽  
Broad Host Range ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Type Iv

ABSTRACT Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

scholarly journals Mycobacterium tuberculosis virulence-regulator PhoP interacts with alternative sigma factor SigE during acid-stress response

2017 ◽  
Vol 104 (3) ◽  
pp. 400-411 ◽  
Author(s):  
Roohi Bansal ◽  
Vijjamarri Anil Kumar ◽  
Ritesh Rajesh Sevalkar ◽  
Prabhat Ranjan Singh ◽  
Dibyendu Sarkar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stress Response ◽  
Sigma Factor ◽  
Acid Stress ◽  
Alternative Sigma Factor ◽  
Acid Stress Response ◽  
Virulence Regulator

scholarly journals Hydrogen Peroxide-Mediated Killing of Caenorhabditis elegans by Streptococcus pyogenes

2002 ◽  
Vol 70 (9) ◽  
pp. 5202-5207 ◽  
Author(s):  
W. T. M. Jansen ◽  
M. Bolm ◽  
R. Balling ◽  
G. S. Chhatwal ◽  
R. Schnabel
Keyword(s):  
Hydrogen Peroxide ◽  
Caenorhabditis Elegans ◽  
Streptococcus Pyogenes ◽  
Wide Spectrum ◽  
Model Organism ◽  
Common Mechanism ◽  
Gram Positive ◽  
C Elegans ◽  
Gram Positive Bacteria ◽  
Serovar Typhimurium

ABSTRACT Caenorhabditis elegans is currently introduced as a new, facile, and cheap model organism to study the pathogenesis of gram-negative bacteria such as Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium. The mechanisms of killing involve either diffusible exotoxins or infection-like processes. Recently, it was shown that also some gram-positive bacteria kill C. elegans, although the precise mechanisms of killing remained open. We examined C. elegans as a pathogenesis model for the gram-positive bacterium Streptococcus pyogenes, a major human pathogen capable of causing a wide spectrum of diseases. We demonstrate that S. pyogenes kills C. elegans, both on solid and in liquid medium. Unlike P. aeruginosa and S. enterica serovar Typhimurium, the killing by S. pyogenes is solely mediated by hydrogen peroxide. Killing required live streptococci; the killing capacity depends on the amount of hydrogen peroxide produced, and killing can be inhibited by catalase. Major exotoxins of S. pyogenes are not involved in the killing process as confirmed by using specific toxin inhibitors and knockout mutants. Moreover, no accumulation of S. pyogenes in C. elegans is observed, which excludes the involvement of infection-like processes. Preliminary results show that S. pneumoniae can also kill C. elegans by hydrogen peroxide production. Hydrogen peroxide-mediated killing might represent a common mechanism by which gram-positive, catalase-negative pathogens kill C. elegans.

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