Role of yoaE Gene Regulated by CpxR in the Survival of Salmonella enterica Serovar Enteritidis in Antibacterial Egg White

ABSTRACT The survival ability of Salmonella enterica serovar Enteritidis in antibacterial egg white is an important factor leading to Salmonella outbreaks through eggs and egg products. In this study, the role of the gene yoaE, encoding an inner membrane protein, in the survival of Salmonella Enteritidis in egg white, and its transcriptional regulation by CpxR were investigated. Quantitative reverse transcription-PCR (RT-qPCR) results showed that the yoaE gene expression was upregulated 35-fold after exposure to egg white for 4 h compared to that in M9FeS medium, and the deletion of yoaE (ΔyoaE) dramatically decreased the survival rate of bacteria in egg white to less than 1% of the wild type (WT) and the complementary strain at both 37 and 20°C, indicating that yoaE was essential for bacteria to survive in egg white. Furthermore, the ΔyoaE strain was sensitive to a 3-kDa ultrafiltration matrix of egg white because of its high pH and antimicrobial peptide components. Putative conserved binding sites for the envelope stress response regulator CpxR were found in the yoaE promoter region. In vivo, the RT-qPCR assay results showed that the upregulation of yoaE in a ΔcpxR strain in egg white was 1/5 that of the WT. In vitro, results from DNase I footprinting and electrophoretic mobility shift assays further demonstrated that CpxR could directly bind to the yoaE promoter region, and a specific CpxR binding sequence was identified. In conclusion, it was shown for the first time that CpxR positively regulated the transcription of yoaE, which was indispensable for survival of Salmonella Enteritidis in egg white. IMPORTANCE Salmonella enterica serovar Enteritidis is the predominant Salmonella serotype that causes human salmonellosis mainly through contaminated chicken eggs or egg products and has been a global public health threat. The spread and frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival in eggs, despite the antibacterial properties of egg white. Research on the survival mechanisms of S. Enteritidis in egg white will help develop effective strategies to control the contamination of eggs by this Salmonella serotype and help further elucidate the complex antibacterial mechanisms of egg white. This study revealed the importance of yoaE, a gene with unknown function, on the survival of S. Enteritidis in egg white, as well as its transcriptional regulation by CpxR. Our work provides the basis to reveal the mechanisms of survival of S. Enteritidis in egg white and the specific function of the yoaE gene.

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Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05006-14 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5357-5365 ◽

Cited By ~ 36

Author(s):

Hilde Smith ◽

Alex Bossers ◽

Frank Harders ◽

Guanghui Wu ◽

Neil Woodford ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Phylogenetic Trees ◽

Functional Study ◽

Content Type ◽

Gene Associations ◽

Genes Encoding ◽

Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

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RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00640-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3666-3675 ◽

Cited By ~ 13

Author(s):

Mei G. Lei ◽

Chia Y. Lee

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factor ◽

Transcriptional Activation ◽

Promoter Region ◽

Mobility Shift ◽

Content Type ◽

Virulence Regulation ◽

Important Virulence Factor ◽

Dna Fragment ◽

Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

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Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

Clinical and Vaccine Immunology ◽

10.1128/cvi.00289-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1491-1498 ◽

Cited By ~ 36

Author(s):

Estela Trebicka ◽

Susan Jacob ◽

Waheed Pirzai ◽

Bryan P. Hurley ◽

Bobby J. Cherayil

Keyword(s):

United States ◽

Bactericidal Activity ◽

Salmonella Enterica ◽

The United States ◽

Healthy Children ◽

Healthy Individuals ◽

Serum Samples ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTRecent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection withSalmonella entericaserovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity againstS. Typhimurium in a standardin vitroassay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) fromS. Typhimurium but not fromEscherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing ofS. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing ofS. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against anS. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

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Flagellin from Recombinant Attenuated Salmonella enterica Serovar Typhimurium Reveals a Fundamental Role in Chicken Innate Immunity

Clinical and Vaccine Immunology ◽

10.1128/cvi.05569-11 ◽

2012 ◽

Vol 19 (3) ◽

pp. 304-312 ◽

Cited By ~ 6

Author(s):

Zhiming Pan ◽

Qiuxia Cong ◽

Shizhong Geng ◽

Qiang Fang ◽

Xilong Kang ◽

...

Keyword(s):

Innate Immunity ◽

Immune Responses ◽

Salmonella Enterica ◽

Rational Design ◽

Bacterial Load ◽

Innate Immune ◽

Innate Immune Responses ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTRecombinant attenuatedSalmonellavaccines have been extensively studied, with a focus on eliciting specific immune responses against foreign antigens. However, very little is known about the innate immune responses, particularly the role of flagellin, in the induction of innate immunity triggered by recombinant attenuatedSalmonellain chickens. In the present report, we describe twoSalmonella entericaserovar Typhimurium vaccine strains, wild-type (WT) or flagellin-deficient (flhD)Salmonella, both expressing the fusion protein (F) gene of Newcastle disease virus. We examined the bacterial load and spatiotemporal kinetics of expression of inflammatory cytokine, chemokine, and Toll-like receptor 5 (TLR5) genes in the cecum, spleen, liver, and heterophils following oral immunization of chickens with the twoSalmonellastrains. TheflhDmutant exhibited an enhanced ability to establish systemic infection compared to the WT. In contrast, the WT strain induced higher levels of interleukin-1β (IL-1β), CXCLi2, and TLR5 mRNAs in cecum, the spleen, and the heterophils than theflhDmutant at different times postinfection. Collectively, the present data reveal a fundamental role of flagellin in the innate immune responses induced by recombinant attenuatedSalmonellavaccines in chickens that should be considered for the rational design of novel vaccines for poultry.

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The H2O2 Stress-Responsive Regulator PerR Positively Regulates srfA Expression in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.187.19.6659-6667.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6659-6667 ◽

Cited By ~ 45

Author(s):

Kentaro Hayashi ◽

Taku Ohsawa ◽

Kazuo Kobayashi ◽

Naotake Ogasawara ◽

Mitsuo Ogura

Keyword(s):

Bacillus Subtilis ◽

Promoter Region ◽

Feeding Assay ◽

Mobility Shift ◽

Genetic Competence ◽

Upstream Promoter ◽

Upstream Promoter Region ◽

Fusion Analysis ◽

Gel Mobility Shift

ABSTRACT srfA is an operon required for the synthesis of surfactin and the development of genetic competence in Bacillus subtilis. We observed that the expression of srfA is downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors in srfA expression. Our DNA microarray analysis revealed that the H2O2 stress-responsive regulator PerR is required for srfA expression. This was confirmed by lacZ fusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR in srfA expression is independent of other known regulators of srfA expression, namely, comQXP, rapC, and spx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region of srfA. A transcriptional srfA-lacZ fusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positive cis elements for srfA transcription.

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Protective Role of Akt2 in Salmonella enterica Serovar Typhimurium-Induced Gastroenterocolitis

Infection and Immunity ◽

10.1128/iai.01235-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2554-2566 ◽

Cited By ~ 15

Author(s):

Winnie W. S. Kum ◽

Bernard C. Lo ◽

Hong B. Yu ◽

B. Brett Finlay

Keyword(s):

Salmonella Enterica ◽

Annexin V ◽

Immunohistochemical Analysis ◽

Neutrophil Infiltration ◽

Protective Role ◽

Necrosis Factor Alpha ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTTheSalmonellaeffector protein SopB has previously been shown to induce activation of Akt and protect epithelial cells from apoptosisin vitro. To characterize the role of Akt2 in host defense againstSalmonella entericaserovar Typhimurium infection, wild-type (WT) mice and mice lacking Akt2 (Akt2 knockout [KO] mice) were infected using aSalmonellaacute gastroenteritis model. Infected Akt2 KO mice showed a more pronounced morbidity and mortality associated with higher bacterial loads in the intestines and elevated levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1, in the colons at 1 day postinfection compared to those shown in WT mice. Histopathological assessment and immunohistochemical analysis of cecal sections at 1 day postinfection revealed more severe inflammation and higher levels of neutrophil infiltration in the ceca of Akt2 KO mice. Flow cytometry analysis further confirmed an increase in the recruitment of Gr-1+CD11b+neutrophils and F4/80+CD11b+macrophages in the intestines of infected Akt2 KO mice. Additionally, enhanced levels of annexin V+and terminal transferase dUTP nick end labeling-positive (TUNEL+) apoptotic cells in the intestines of infected Akt2 KO mice were also observed, indicating that Akt2 plays an essential role in protection against apoptosis. Finally, the differences in bacterial loads and cecal inflammation in WT and Akt2 KO mice infected with WTSalmonellawere abolished when these mice were infected with thesopBdeletion mutant, indicating that SopB may play a role in protecting the mice fromSalmonellainfection through the activation of Akt2. These data demonstrate a definitive phenotypic abnormality in the innate response in mice lacking Akt2, underscoring the important protective role of Akt2 inSalmonellainfection.

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Multiple Transcription-Activating Sequences Regulate the RsmZ Regulatory Small RNA of Pseudomonas brassicacearum

Journal of Bacteriology ◽

10.1128/jb.00408-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4888-4893 ◽

Cited By ~ 2

Author(s):

D. Lalaouna ◽

S. Fochesato ◽

M. Barakat ◽

P. Ortet ◽

W. Achouak

Keyword(s):

Small Rna ◽

Promoter Region ◽

Promoter Element ◽

Content Type ◽

Consensus Motifs ◽

Encoding Gene ◽

Pseudomonas Brassicacearum ◽

Activation Sequence

ABSTRACTThemutS-rpoSregion is known to be a highly polymorphic segment of the chromosome owing to horizontal gene transfer and evolutionary processes. InPseudomonas,mutS-fdxA-rsmZ-rpoSorganization is highly conserved, as well as the promoter region of the RsmZ small RNA (sRNA)-encoding gene. One exception to this conservation is inPseudomonas brassicacearum, where a 308-nucleotide (nt) sequence, predicted to form a hairpin structure in single-stranded DNA (ssDNA), is inserted between therpoSandrsmZgenes. Using MEME software, we identified nine consensus motifs in thersmZpromoter region of 16 sequencedPseudomonasgenomes. We observed that an upstream activation sequence (UAS) and an M1 motif (located between the −10 promoter element and the UAS) are shared among examinedPseudomonasgenomes. A third motif, the M2 motif, is localized within the coding sequence of therpoSgene. Constructs fusing the different identified motifs to thelacZreporter were produced. Ourin vivoanalysis of thersmZ-activating elements indicates that the palindromic UAS located 180 bp upstream of thersmZtranscriptional start inP. brassicacearumNFM 421 is essential, but not sufficient, for fullrsmZexpression. Here, we demonstrate a role for the three motifs in the activation of thersmZgene, and we hypothesize the role of additional transcriptional factors, along with the DNA structuring role of the hairpin in the complex network controlling the expression ofrsmZ.

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iac Gene Expression in the Indole-3-Acetic Acid-Degrading Soil Bacterium Enterobacter soli LF7

Applied and Environmental Microbiology ◽

10.1128/aem.01057-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 6

Author(s):

Isaac V. Greenhut ◽

Beryl L. Slezak ◽

Johan H. J. Leveau

Keyword(s):

Gene Expression ◽

Acetic Acid ◽

Promoter Region ◽

Deletion Mutant ◽

Plant Hormone ◽

Major Facilitator Superfamily ◽

Soil Bacterium ◽

Content Type ◽

Indole 3 Acetic Acid

ABSTRACT We show for soil bacterium Enterobacter soli LF7 that the possession of an indole-3-acetic acid catabolic (iac) gene cluster is causatively linked to the ability to utilize the plant hormone indole-3-acetic acid (IAA) as a carbon and energy source. Genome-wide transcriptional profiling by mRNA sequencing revealed that these iac genes, chromosomally arranged as iacHABICDEFG and coding for the transformation of IAA to catechol, were the most highly induced (>29-fold) among the relatively few (<1%) differentially expressed genes in response to IAA. Also highly induced and immediately downstream of the iac cluster were genes for a major facilitator superfamily protein (mfs) and enzymes of the β-ketoadipate pathway (pcaIJD-catBCA), which channels catechol into central metabolism. This entire iacHABICDEFG-mfs-pcaIJD-catBCA gene set was constitutively expressed in an iacR deletion mutant, confirming the role of iacR, annotated as coding for a MarR-type regulator and located upstream of iacH, as a repressor of iac gene expression. In E. soli LF7 carrying the DNA region upstream of iacH fused to a promoterless gfp gene, green fluorescence accumulated in response to IAA at concentrations as low as 1.6 μM. The iacH promoter region also responded to chlorinated IAA, but not other aromatics tested, indicating a narrow substrate specificity. In an iacR deletion mutant, gfp expression from the iacH promoter region was constitutive, consistent with the predicted role of iacR as a repressor. A deletion analysis revealed putative −35/−10 promoter sequences upstream of iacH, as well as a possible binding site for the IacR repressor. IMPORTANCE Bacterial iac genes code for the enzymatic conversion of the plant hormone indole-3-acetic acid (IAA) to catechol. Here, we demonstrate that the iac genes of soil bacterium Enterobacter soli LF7 enable growth on IAA by coarrangement and coexpression with a set of pca and cat genes that code for complete conversion of catechol to central metabolites. This work contributes in a number of novel and significant ways to our understanding of iac gene biology in bacteria from (non-)plant environments. More specifically, we show that LF7's response to IAA involves derepression of the MarR-type transcriptional regulator IacR, which is quite fast (less than 25 min upon IAA exposure), highly specific (only in response to IAA and chlorinated IAA, and with few genes other than iac, cat, and pca induced), relatively sensitive (low micromolar range), and seemingly tailored to exploit IAA as a source of carbon and energy.

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Extensive Genetic Variability Linked to IS26Insertions in thefljBPromoter Region of Atypical Monophasic Variants of Salmonella enterica Serovar Typhimurium

Applied and Environmental Microbiology ◽

10.1128/aem.00270-15 ◽

2015 ◽

Vol 81 (9) ◽

pp. 3169-3175 ◽

Cited By ~ 11

Author(s):

Cécile Boland ◽

Sophie Bertrand ◽

Wesley Mattheus ◽

Katelijne Dierick ◽

Vicky Jasson ◽

...

Keyword(s):

Salmonella Enterica ◽

Promoter Region ◽

Variable Number Tandem Repeat ◽

Genetic Alterations ◽

Variable Number ◽

Genomic Region ◽

Phase 2 ◽

Pathogenic Potential ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTFifty-nine monophasicSalmonella entericaserovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:− and shown to harbor anfljBcoding sequence. The genetic differences between these strains and phenotypically biphasicSalmonellaTyphimurium were analyzed through PCR and DNA sequencing. Genetic alterations in thefljBpromoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying thefljBpromoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasicSalmonellaTyphimurium strain could be observed. Next-generation sequencing of one representative isolate affected in thefljBpromoter region revealed a 26-kb IS26composite transposon insertion along with a local genomic rearrangement. Several other IS26element-mediated alterations of this genomic region were observed. This group of monophasicSalmonellaTyphimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasicSalmonellaTyphimurium ancestors.

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Bacillus anthracis Virulence Regulator AtxA Binds Specifically to the pagA Promoter Region

Journal of Bacteriology ◽

10.1128/jb.00569-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 1

Author(s):

Rita M. McCall ◽

Mary E. Sievers ◽

Rasem Fattah ◽

Rodolfo Ghirlando ◽

Andrei P. Pomerantsev ◽

...

Keyword(s):

Gene Regulation ◽

Bacillus Anthracis ◽

Promoter Region ◽

Virulence Gene ◽

Anthrax Toxin ◽

Content Type ◽

Polymerase Binding

ABSTRACT Anthrax toxin activator (AtxA) is the master virulence gene regulator of Bacillus anthracis. It regulates genes on the chromosome as well as the pXO1 and pXO2 plasmids. It is not clear how AtxA regulates these genes, and direct binding of AtxA to its targets has not been shown. It has been previously suggested that AtxA and other proteins in the Mga/AtxA global transcriptional regulators family bind to the curvature of their DNA targets, although this has never been experimentally proven. Using electrophoretic mobility shift assays, we demonstrate that AtxA binds directly to the promoter region of pagA upstream of the RNA polymerase binding site. We also demonstrate that in vitro, CO2 appears to have no role in AtxA binding. However, phosphomimetic and phosphoablative substitutions in the phosphotransferase system (PTS) regulation domains (PRDs) do appear to influence AtxA binding and pagA regulation. In silico, in vitro, and in vivo analyses demonstrate that one of two hypothesized stem-loops located upstream of the RNA polymerase binding site in the pagA promoter region is important for AtxA binding in vitro and pagA regulation in vivo. Our study clarifies the mechanism by which AtxA interacts with one of its targets. IMPORTANCE Anthrax toxin activator (AtxA) regulates the major virulence genes in Bacillus anthracis. The bacterium produces the anthrax toxins, and understanding the mechanism of toxin production may facilitate the development of therapeutics for B. anthracis infection. Since the discovery of AtxA 25 years ago, the mechanism by which it regulates its targets has largely remained a mystery. Here, we provide evidence that AtxA binds to the promoter region of the pagA gene encoding the main central protective antigen (PA) component of the anthrax toxin. These data suggest that AtxA binding plays a direct role in gene regulation. Our work also assists in clarifying the role of CO2 in AtxA’s gene regulation and provides more evidence for the role of AtxA phosphorylation in virulence gene regulation.

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