Project description:BACKGROUND: An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. RESULTS: We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. CONCLUSIONS: We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.
Project description:Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, ?4)-?-d-ManpNAc-(1 ? 4)-?-d-GlcpNAc-(1 ? 6)-?-d-GlcpNAc-(1?. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-?-d-ManpNAc-(1 ? 4)-[3-O-acetyl]-?-d-GlcpNAc-(1 ? 6)-?-d-GlcpNH(2)-(1?.
Project description:The goal of this study is the discovery of (a) meaningful phylogenomic relationships among members of this B. cereus/B. anthracis group, and (b) reliable gene-phenotype associations, e.g. recognition of links between genomic traits and the ability of certain strains to cause various forms of disease. We also tried to elucidate genome evolution aspects that may lead to the emergence of variants that are capable (or have the potential) of causing anthrax-like disease. This large-scale comparative genomics approach is unprecedented for this taxonomic group. Dr. A. Hoffmaster (CDC) provided the PFGRC with 73 B. cereus and B. anthracis isolates from the CDC culture collection. Of these, 27 were isolated from patients with severe or systemic disease; ten isolates of this group were obtained from patients (welding factory workers) with anthrax-like disease or from the environment near their workplace. Another set of 26 represented isolates from food-born illnesses. Of the 26 gastrointestinal disease isolates (GIDI), 10 were obtained from patients with diarrhea, whereas another set of 10 had been shown to harbor the emetic (vomit) toxin gene by PCR. The rest of the group consisted of 20 isolates with various phenotypes. All strains were screened for their genomic content using the B. cereus/B. anthracis species microarray. Seventy-three query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Dye-swap experiments were performed with all the 73 strains on both chipA and chipB of the microarray, for a total of four or more hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.
Project description:Investigation of whole genome expression level changes in Bacillus anthracis Sterne deltaClpX mutant compared to the wild-type strain after growth in nutrient rich media. The deltaClpX mutant used in this study is described in McGillivray et al. 2009. ClpX Protease Contributes to Antimicrobial Peptide Resistance and Virulence Phenotypes of Bacillus anthracis. Journal of Innate Immunity 1(5): 494-506.