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.Coenzyme A (CoASH) (20) is the major low-molecularweight thiol in Bacillus anthracis (25); the tripeptide thiol glutathione (8) is absent in all species of Bacillus analyzed to date (23,24,32 (25) have demonstrated a strong correlation among the absence of glutathione biosynthesis, the absence of the type I PanK, and the presence of the type III enzyme for 14 phylogenetic classes of bacteria.In their transcriptional profiling of the B. anthracis life cycle, in which five distinct temporal waves of gene expression were identified from germination through sporulation, Bergman et al. (4) showed that the coaX, coaBC (BA4007), and coaD (BA4139) genes encoding the first three enzymes in the Pan3CoASH pathway (20) are upregulated in waves I and II. The coaX gene was also upregulated more than twofold between 1 and 2 h postinfection within host macrophages (3); in this respect (4), PanK Ba may be particularly significant as a potential target for the design of therapeutically useful molecules. CoASH biosynthesis has very recently been reviewed as an antimicrobial drug target (31); crystal structures are now available for PanK Ba (25) and the type III PanKs from Pseudomonas aeruginosa (15) and Thermotoga maritima (35, 36), and they include complexes with Pan and ADP and with the 4Ј-phosphopantothenate product. These have led to the identification of new motifs for the Pan-binding pocket and suggest, based on differences in the binding modes for both Pan and ATP substrates (relative to the type II human PanK) (14), potential modes of design for new inhibitors specifically targeting the type III enzymes.As suggested by the absence of glutathione, CoASH also functions in the thiol-disulfide redox biology of B. anthracis (25); this scheme includes an NAD(P)H-dependent CoA-disulfide reductase (BACoADR) that maintains the reduced intracellular state of CoASH in vegetative cells via an enzymatic . This is quite distinct from the system described for B. subtilis, which has both type I and type III PanKs (6, 37) but lacks CoA-disulfide reductase (34). While the recent report of Hochgräfe et al. (13) supports the conclusion that S-thiolation by Cys represents a general, reversible mechanism for the protection of protein-SH groups during disulfide stress in B. subtilis, earlier studies with Bacillus megaterium linked CoA-disulfide reductase (32) with the reduction of spore protein-SSCoA (soluble proteins S-thiolated with CoASH) early in germination. These protein-SSCoA mixed disulfide forms account for ϳ45% of the total CoASH in B. megaterium spores (30).Bioinformatics analyses of the PanK Ba locus (26) indicated that coaX might be linked with the hslO and cysK-1 genes (BA0066 and BA0067, r...

Essentiality and functional analysis of type I and type III pantothenate kinases of Mycobacterium tuberculosis

“…This has been found to be true in B. subtilis, wherein it has been demonstrated that the presence of either CoaA or CoaX permits the cell to be viable (Yocum & Patterson, 2004). However, in a related bacterium, B. anthracis, which possesses type II and III isoforms of pantothenate kinase (Yang et al 2006), the coaX gene was found to be essential even in the presence of type II isoform of pantothenate kinase (Paige et al, 2008), which led the authors to propose CoaX as a drug target for developing inhibitors (Paige et al, 2008). Whether the type II pantothenate kinase of B. anthracis is also essential or not is not clear at this moment.…”

“…1, 6, genomic DNA; 2, 7, RT template; 3, 8, RT-minus template; 4, 9, no template; 5, molecular size marker. (Dunn & Snell 1979;Vallari & Rock, 1987;Wood & Friedman, 2000;Liberati et al, 2006: Paige et al, 2008, it can be speculated that either of the two kinase coding genes (coaA or coaX) could potentially be capable of providing the essential enzymic function for the survival of M. tuberculosis. In order to prove whether either of them could keep the cells alive in the absence of other isoforms (as in B. subtilis) or only one is essential even in the presence of the other isoform (as in B. anthracis), we attempted to inactivate the coaA and coaX genes of M. tuberculosis individually.…”

“…The coaX gene has been shown to be essential for survival of B. anthracis, P. aeruginosa and Bordetella pertussis (Wood & Friedman, 2000;Liberati et al, 2006;Paige et al, 2008). In order to assess whether CoaX is essential for the survival of M. tuberculosis, we decided to inactivate the coaX gene by homologous recombination using the strategy applied for coaA KO described above.…”

“…However, in B. anthracis, the coaX gene has been found to be essential even in the presence of a gene encoding a type II pantothenate kinase, indicating that their functions are not interchangeable in this organism. (Paige et al, 2008). A homologue of CoaX in Bordetella pertussis, BvgS has been shown to be essential for viability (Wood & Friedman, 2000).…”

Genetic Manipulation Methods inBacillus anthracis

“…For this reason, generation of stable mutants by allelic exchange is the preferred method of isolating mutants in B. anthracis . This method of isolating single crossover insertion of plasmids has been used to examine whether particular genes of interest are essential (Paige et al, 2008 ); the inability to isolate viable cells when both levels of selection are administered is consistent with the conclusion that the genes in question are essential for growth. These protocols have also been adapted for regulatory studies of genes in B. anthracis .…”