The type III pantothenate kinase encoded by coaX is essential for growth of Bacillus anthracis.
Ontology highlight
ABSTRACT:
SUBMITTER: Paige C
PROVIDER: S-EPMC2546792 | biostudies-literature | 2008 Sep
REPOSITORIES: biostudies-literature
Publications
The type III pantothenate kinase encoded by coaX is essential for growth of Bacillus anthracis.
Paige Carleitta C Reid Sean D SD Hanna Philip C PC Claiborne Al A
Journal of bacteriology 20080718 18
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. ...[more]
Similar Datasets
Project description:Antibiotics with novel mechanisms of action are desperately needed to combat the increasing rates of multidrug-resistant infections. Bacterial pantothenate kinase (PanK) has emerged as a target of interest to cut off the biosynthesis of coenzyme A. Herein we report the results of an in vitro high-throughput screen of over 10 000 small molecules against Bacillus anthracis PanK, as well as a follow-up screen of hits against PanK isolated from Pseudomonas aeruginosa and Burkholderia cenocepacia. Nine hits are structurally categorized and analyzed to set the stage for future drug development.
| S-EPMC6456334 | biostudies-literature
Project description:Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-89]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 A, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.
| S-EPMC2613803 | biostudies-literature
Project description:Bacillus anthracis Sterne cured of the pXO1 plasmid had enhanced secreted protease activity during the postexponential phase but no change in hemolytic or lecithinase activities. A zymogen profile revealed at least six proteases, including serine, metal, and perhaps cysteine types. There were similar amounts of protease secreted by the closely related species Bacillus cereus and Bacillus thuringiensis, but the patterns differed. Among the pXO1 plasmid-encoded proteins, there is a tetratricopeptide protein designated Cot43 that is related to the Rap proteins of Bacillus subtilis and the PlcR pleiotropic regulator of secreted enzymes and toxins in B. thuringiensis. A disruption of the cot43 gene resulted in overproduction of several proteases to a somewhat greater extent than in the plasmid-cured strain. Transformation of either of these strains with a clone of the cot43 gene resulted in the inhibition of accumulation of some of the proteases and induction of at least one. On the basis of lacZ fusions, transcription of the cot43 gene increased in late exponential cells at the time of protease accumulation. The expression of lacZ fusions to the upstream regions of two B. anthracis extracellular protease genes was greater in the strain with the disruption of cot43 than in the Sterne strain, indicating regulation at the level of transcription. In B. anthracis, a pXO1 plasmid-encoded protein directly modulates or indirectly regulates the transcription of genes for several chromosomally encoded extracellular proteases.
| S-EPMC1082805 | biostudies-literature
Project description:Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.
| S-EPMC1595385 | biostudies-literature
Project description:Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK(Tm)), solved at 2.0-A resolution. The structure of PanK(Tm) reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK(Tm) active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK -- which is a member of the "P-loop kinase"superfamily -- this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.
| S-EPMC1540032 | biostudies-literature
Project description:A Bacillus anthracis virulence plasmid-encoded peptidoglycan hydrolase (AmiA) with sequence similarity to N-acetylmuramoyl-L-alanine amidases hydrolyzes peptidoglycan independently of cell wall binding. Residues H341, E355, H415, and E486 are absolutely required for catalysis. Many AmiA paralogs are fused to different sorting signals, suggesting that these modular proteins result from domain shuffling.
| S-EPMC134760 | biostudies-literature
Project description:In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention.
| S-EPMC2582132 | biostudies-literature
Project description:Purpose: Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been extensively studied in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by the virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. We designated the sRNAs as as “XrrA” and “XrrB” (for pXO1-encoded regulatory RNA). We constructed deletion mutants ∆xrrA, ∆xrrB, and ∆xrrA∆xrrB in an Ames strain background and compared gene expression of these mutants to that of the Ames-derived parent strain using RNA-seq. Methods: Total RNA from Ames strains UTA37 (Parent), UTA38 (∆xrrB), UTA39 (∆xrrA), UTA41 (∆xrrA∆xrrB) was isolated from cultures grown in CA-CO2 in triplicate, using saturated phenol: chloroform extraction. One microgram of pure and quality-checked RNA from each sample was subjected to Illumina sequencing by synthesis using a NextSeq550 sequencer. Sequencing by synthesis was performed to generate 75bp paired-end reads. Two 130M-read sequencing runs (Run1 and Run2) were performed and an average of 31M reads per sample were obtained. Results: All bioinformatic analysis was performed using the publicly available Galaxy web resource (https://usegalaxy.org/). Comparison of the transcriptomes of a virulent Ames-derived strain to isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Deletion of xrrA resulted in 50 transcripts showing a ≥ 4.0-fold change in expression compared to the parent strain. In contrast, deletion of xrrB led to one transcript having a ≥ 4.0-fold change in expression compared to the parent strain. In the ∆xrrA∆xrrB mutant, levels of 116 transcripts were affected with a fold-change of ≥ 4.0. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Conclusions: In this work, we provide experimental evidence for sRNA-mediated regulation in the mammalian pathogen B. anthracis. XrrA and XrrB are the first regulatory RNAs examined in B. anthracis and are shown here to have multiple effects on gene expression in this pathogen. The data suggest distinct regulatory roles for XrrA, as well as some functional overlap between XrrA and XrrB.
2021-02-02 | GSE152356 | GEO
Project description:BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.
| S-EPMC3133050 | biostudies-literature
Project description:Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K (m) and V (max) values of 33 microM and 48 micromol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was approximately 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC(50) value of 10 microM, while other nucleosides--L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine--were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.
| S-EPMC2525521 | biostudies-literature