Dual Promoters Control Expression of the Bacillus anthracis Virulence Factor AtxA

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Journal of bacteriology (Impact Factor: 2.81). 09/2008; 190(19):6483-92. DOI: 10.1128/JB.00766-08
Source: PubMed


The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein
whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription
of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T.
M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have −10 and −35 consensus
sequences compatible with recognition by σA-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity
and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the
level of AtxA in the cell.

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Available from: Tatsuya Fukushima, Nov 22, 2014
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    • "AtxA is a transcriptional regulator containing two DNA binding domains in the N-terminal region [11]. At the transcriptional level, production of atxA mRNA is upregulated by temperatures close to 37°C [16], repressed by the transition-state regulator AbrB [17,18], and impacted by the cellular redox potential [19], but these changes are all quite modest. At the post-translational level, AtxA is regulated via the phosphorylation of two histidine residues [20] and an unknown mechanism requiring the presence of the global regulator CodY [21,22]. "
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    ABSTRACT: Upon infection of a mammalian host, Bacillus anthracis responds to host cues, and particularly to elevated temperature (37[degree sign]C) and bicarbonate/CO2 concentrations, with increased expression of virulence factors that include the anthrax toxins and extracellular capsular layer. This response requires the presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To better understand the genetic basis of this response, we utilized a controlled in vitro system and Next Generation sequencing to determine and compare RNA expression profiles of the parental strain and an isogenic AtxA-deficient strain in a 2 x 2 factorial design with growth environments containing or lacking carbon dioxide. We found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. A majority of genes on the virulence plasmid pXO1 that are regulated by the presence of either CO2 or AtxA separately are also regulated synergistically in the presence of both. These results also elucidate novel pXO1-encoded small RNAs that are associated with virulence conditions.
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    • "D. The resB deletion affects only the P1 promoter of atxA. The 34F2 and 34F2△resB strains were transformed with different atxA-lacZ fusion constructs in pTCV-lac (Bongiorni et al., 2008) and β-galactosidase activity was measured. Symbols: -▪-34F2/pAtxA10; -□- 34F2△resB/pAtxA10; -▲-34F2/pAtxA12; -△-34F2△resB/pAtxA12; -◆-34F2/ pAtxA20; -◇-34F2△resB/pAtxA20. "
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    ABSTRACT: Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c-type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c-type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.
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