scholarly journals The Alternative Sigma Factor σH Is Required for Toxin Gene Expression by Bacillus anthracis

2006 ◽  
Vol 189 (5) ◽  
pp. 1874-1883 ◽  
Author(s):  
Maria Hadjifrangiskou ◽  
Yahua Chen ◽  
Theresa M. Koehler
Keyword(s):  
Gene Expression ◽  
Bacillus Anthracis ◽  
Sigma Factor ◽  
State Level ◽  
Growth Conditions ◽  
Exponential Phase ◽  
Toxin Gene ◽  
Gene Encoding ◽  
Independent Manner ◽  
Toxin Gene Expression

ABSTRACT Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated CO2, and the trans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH, a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis. In the well-studied B. subtilis system, σH is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. σH positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of σH and AbrB on the expression of atxA.

scholarly journals The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence

2009 ◽  
Vol 77 (10) ◽  
pp. 4437-4445 ◽  
Author(s):  
Willem van Schaik ◽  
Alice Château ◽  
Marie-Agnès Dillies ◽  
Jean-Yves Coppée ◽  
Abraham L. Sonenshein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Anthracis ◽  
Mutant Strain ◽  
Virulence Gene ◽  
Etiologic Agent ◽  
Toxin Gene ◽  
Promoter Regions ◽  
Virulence Gene Expression ◽  
Toxin Gene Expression ◽  
Virulence Regulator

ABSTRACT In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

scholarly journals The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Gene Expression ◽  
Sigma Factor ◽  
Cysteine Residue ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Content Type ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

ABSTRACT Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (OB-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors. IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiTopt) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development.

scholarly journals Environmental Response and Autoregulation of Clostridium difficile TxeR, a Sigma Factor for Toxin Gene Expression

2002 ◽  
Vol 184 (21) ◽  
pp. 5971-5978 ◽  
Author(s):  
Nagraj Mani ◽  
Dena Lyras ◽  
Lisa Barroso ◽  
Pauline Howarth ◽  
Tracy Wilkins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Growth Phase ◽  
Sigma Factor ◽  
Cellular Growth ◽  
Broth Culture ◽  
Exponential Growth Phase ◽  
Toxin Gene ◽  
Toxin Genes ◽  
Toxin Gene Expression

ABSTRACT TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli.

scholarly journals The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

2019 ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Sigma Factor ◽  
Negative Regulator ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
N Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

AbstractClostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

scholarly journals RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Content Type ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

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