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ABSTRACT Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of mammalian hosts. The metabolic attributes of B. anthracis that permit rapid growth in multiple mammalian tissues have not been established. We posit that branched-chain amino acid (BCAA) (isoleucine, leucine, and valine) metabolism is key to B. anthracis pathogenesis. Increasing evidence indicates the relationships between B. anthracis virulence and the expression of BCAA-related genes. The expression of some BCAA-related genes is altered during culture in bovine blood in vitro, and the bacterium exhibits valine auxotrophy in a blood serum mimic medium. Transcriptome analyses have revealed that the virulence regulator AtxA, which positively affects the expression of the anthrax toxin and capsule genes, negatively regulates genes predicted to be associated with BCAA biosynthesis and transport. Here, we show that B. anthracis growth in defined medium is severely restricted in the absence of exogenous BCAAs, indicating that BCAA transport is required for optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-type BCAA transporters. Three transporters are associated with isoleucine and valine transport, and the deletion of one, BrnQ3, attenuates virulence in a murine model for anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy acid dehydratase, an enzyme essential for BCAA synthesis, exhibits unperturbed growth when cultured in medium containing BCAAs but is highly attenuated in the murine model. Finally, our data show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a model in which BCAAs serve as a signal for virulence gene expression. IMPORTANCE Infection with B. anthracis can result in systemic disease with large numbers of the bacterium in multiple tissues. We found that branched-chain amino acid (BCAA) synthesis is insufficient for the robust growth of B. anthracis; access to BCAAs is necessary for the proliferation of the pathogen during culture and during infection in a murine model for anthrax. B. anthracis produces an unusually large repertoire of BCAA-related transporters. We identified three isoleucine/valine transporters with partial functional redundancy during culture. The deletion of one of these transporters, BrnQ3, resulted in attenuated virulence. Interestingly, a BCAA biosynthesis mutant grew well in medium containing BCAAs but, like BrnQ3, was attenuated for virulence. These results suggest that BCAAs are limiting in multiple niches during infection and further our understanding of the nutritional requirements of this important pathogen.

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.