K-100. The Type III Pantothenate Kinase in Bacillus anthracis Is a Candidate for Therapeutic Intervention Against Anthrax Infection

C. Paige1, S. D. Reid1, P. Hanna2, A. Claiborne1;
1Wake Forest Univ. Sch. of Med., Winston-Salem, NC, 2Univ. of Michigan Sch. of Med., Ann Arbor, MI.

The first committed step in coenzyme A (CoASH) biosynthesis is catalyzed by pantothenate kinase (PanK). Of the three characterized bacterial PanK isoforms, Bacillus anthracis utilizes the type III PanK as its sole functional enzyme in 4´-phosphopantothenate synthesis. The type III PanK (BaPanK) is the first structural gene in a tricistronic operon (coaX-hslO-cysK-1), which also encodes the redox-regulated Hsp33 heat shock protein and cysteine synthase A. Repeated attempts to generate a stable coaX deletion mutant in B. anthracis led only to clones retaining the wild-type locus, suggesting that BaPanK is essential for growth. Therefore, we have generated the conditional coaX mutant that carries a copy of the wild-type coaX-hslO-cysK-1 locus under the control of the IPTG-inducible PSPAC promoter. As anticipated, the mutant displays essentially wild-type growth in BHI plus 50 μM IPTG; we did not, however, anticipate the growth observed ~7 hours post-inoculation in the absence of IPTG. This unexpected result is due to the development of a stable suppressor mutation; a point mutation (G→A) has been identified in the PSPAC promoter, which may be responsible for the transcription observed for coaX (and hslO-cysK-1) genes in the absence of IPTG. Due to polar effects in the conditional mutant, we implemented gene disruption strategies to evaluate whether hslO and/or cysK-1 were essential. We have shown that both cysK-1 and hslO are not essential and therefore, do not contribute the conditional mutant growth phenotype. Thus, evidence to date supports BaPanK as a potential target for therapeutic intervention against anthrax infection.