The type III pantothenate kinase encoded by coaX is essential for growth of Bacillus anthracis

Abstract

In Bacillus anthracis, the novel type III pantothenate kinase (PanK(Ba); encoded by coaX) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional coaX mutant that PanK(Ba) is an essential enzyme, thus contributing to its validation as a new antimicrobial target.

FIG. 1.

coaX is part of a tricistronic operon during the exponential growth of B. anthracis Sterne. (A) Genomic contexts (26) for coaX in B. anthracis Sterne (top) and B. subtilis (bottom). Primer sets 1 to 5 for endpoint RT-PCR (arrows) are indicated above the B. anthracis open reading frames and correspond as follows: 1, ftsH-coaX; 2, coaX-hslO; 3, hslO-cysK-1; 4, cysK-1 inner; and 5, cysK-1-pabB. (B) Endpoint RT-PCR showing that coaX is transcribed as part of a contiguous transcript during exponential growth. The gel shows contiguous products (at an A₆₀₀ of 0.4 to 0.6) from primer sets 2 to 4 but not from pairs 1 or 5. Controls with genomic DNA and without RT were done for each experiment. (C) Partial sequence of the intergenic region between B. anthracis Sterne ftsH and coaX. Elements of the σ^A-dependent promoter sequence (−35, TG, and −10) are underlined, as is the ribosome binding site. The coaX transcription start site, as determined by 5′ RACE analysis, is indicated by a bent arrow; the start codon (ATG) is boxed.

FIG. 2.

Construction of the COAXd mutant strain and the effect of IPTG addition on growth. (A) At the permissive temperature of 30°C, PCR analyses confirm the presence of the independently replicating pNFd13::coaX′ plasmid (lane 2), the intact chromosomal copy of coaX (lane 3), and the coaX::pNFd13 integrant (lanes 4 and 5). At the restrictive temperature of 39°C, PCR analyses confirm the absence of both the independently replicating plasmid (lane 6) and the intact coaX locus (lane 7), as well as the presence of the chromosomal integrant (lanes 8 and 9). Lane 1 is a 1-kb ladder. The four primer combinations used, both for lanes 2 to 5 and for lanes 6 to 9, were NFd-FOR/NFd-REV, US-FOR/DS-REV (coaX), US-FOR/NFd-REV, and NFd-FOR/DS-REV. Sequences are available on request. (B) Effects of IPTG addition on the B. anthracis Sterne COAXd mutant strain. The COAXd strain, in which the expression of coaX is controlled by P_spac, was grown in the presence of 50 μM IPTG and diluted 40-fold into fresh prewarmed BHI broth with (▪) or without 50 μM IPTG. The wild-type strain (□) is included as a control. At 4 h, different concentrations of IPTG were added (indicated by the arrow) to those cultures inoculated without IPTG. •, COAXd without IPTG; ○, COAXd with 5 μM IPTG; ▵, COAXd with 10 μM IPTG; and ▴, COAXd with 50 μM IPTG. The COAXd(Su) suppressor mutant is responsible for growth observed in the absence of IPTG.

FIG. 3.

Construction of hslO and cysK-1 fusion mutants. Diagnostic PCR confirming the correct integrations of pBKJ236::hslO′ (lanes 2 and 3) and pBKJ236::cysK-1′ (lanes 4 and 5) into hslO and cysK-1 loci, respectively. Lane 1 is a 1-kb ladder. The respective primer combinations used were as follows (sequences available upon request): lane 2, US-FOR/BKJ-REV (hslO); lane 3, BKJ-FOR/DS-REV (hslO); lane 4, US-FOR/BKJ-REV (cysK-1); and lane 5, BKJ-FOR/DS-REV (cysK-1).

FIG. 4.

The COAXd(Su) suppressor mutant carries a single-base substitution within the lac operator and constitutively produces the tricistronic coaX transcript. (A) Multiple alignment of partial sequences for the P_spac promoter regions of pNFd13 (P_spac), the COAXd conditional mutant grown in the presence of IPTG, and the COAXd(Su) suppressor mutant isolated in the absence of IPTG. Four individual colonies were analyzed for both COAXd and COAXd(Su), with internally identical results. The σ^A-dependent promoter and lac operator sequences are boxed. The COAXd(Su) lac operator carries a guanine→adenine mutation in position 5; this position is boxed and indicated with an asterisk. (B) Total RNAs were isolated from COAXd and COAXd(Su) as described for wild-type B. anthracis Sterne, but cultures were grown in the presence and absence, respectively, of IPTG and at 39°C. Samples were analyzed by endpoint RT-PCR using the primer sets described in Fig. 1B. The gels show contiguous coaX transcripts for both samples.