The stringent response of Bacillus anthracis contributes to sporulation but not to virulence

The Gram-positive, spore-forming pathogen Bacillus anthracis is the aetiological agent of anthrax. Its main virulence factors are two toxins and an anti-phagocytic capsule. When B. anthracis is grown in laboratory culture, the highest expression of the anthrax toxin genes occurs during entry into stationary phase, suggesting that nutrient limitation is an environmental cue which induces toxin production. A common bacterial response to starvation is the so-called stringent response, in which the hyperphosphorylated guanosine nucleotide (p)ppGpp is the effector molecule. In Escherichia coli, Bacillus subtilis and other bacteria, accumulation of this molecule leads to down-regulation of stable RNA synthesis and upregulation of the expression of genes involved in survival under nutrient-poor conditions. This study focuses on the stringent response of B. anthracis. We show that in B. anthracis the relA gene is responsible for the synthesis of (p)ppGpp and the stringent down-regulation of stable RNA synthesis upon starvation for the essential amino acids isoleucine, leucine and valine. The deletion of relA did not affect the expression of the virulence gene pagA or virulence in a mouse model of infection. In contrast, spore counts upon growth and sporulation in a defined medium were approximately 10,000-fold lower for the relA deletion mutant than for the parental strain. The contribution of the stringent response to efficient sporulation of B. anthracis is notable, as this suggests that the stringent response may contribute to the persistence of B. anthracis in the natural environment.