cis -Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis

Abstract

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5= and 3= deletion analysis and sitedirected mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A؉T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between ؊13 and ؉36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.

The Two CcdA Proteins of Bacillus anthracis Differentially Affect Virulence Gene Expression and Sporulation

“…This effect, however, is seen only under in vitro conditions that do not induce strong toxin production (10). It was recently reported that manipulation of the atxA promoter to overexpress AtxA did not alter virulence in a murine model of infection (49). While this experiment did not directly replicate the unique atxA expression profile found in the loss of ccdA1 and ccdA2, it does raise questions about the relevance of AtxA overexpression.…”

Regulons and protein–protein interactions of PRD‐containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions

“…Finally, an unknown additional repressor binds to the atxA promoter at a palindromic sequence from – 13 to + 31 relative to the P1 transcription start site. Deletion of this sequence results in a 15‐fold increase in atxA transcription (Dale et al ., ).…”

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

“…In these culture conditions, which are thought to mimic the mammalian host environment, AtxA positively controls transcription of the three structural genes, lef , pagA and cya , for the anthrax toxin proteins, the capsule biosynthetic operon capBCADE , and numerous other genes on the B. anthracis chromosome and plasmids (Bourgogne et al ., ). atxA ‐null mutants are highly attenuated in murine models for anthrax (Dai et al ., ; Dale et al ., ). Thus, this PCVR is viewed as a critical regulator of B. anthracis virulence.…”

“…Transcription of atxA is affected by temperature, redox potential, growth phase and the presence of glucose (Dai and Koehler, ; Saile and Koehler, ; Wilson et al ., ; Chiang et al ., ). The transition state regulator AbrB negatively affects atxA expression (Saile and Koehler, ; Strauch et al ., ), and other trans ‐acting regulators have also been implicated in atxA transcription (Chiang et al ., ; Dale et al ., ). Growth of B. anthracis in media containing bicarbonate in elevated levels of atmospheric CO 2 affects AtxA activity post‐transcriptionally (Koehler et al ., ; Sirard et al ., ; Dai et al ., ; Dai and Koehler, ; Hammerstrom et al ., ).…”