Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant

(BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype.Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation

Comparative Genomics of 2009 Seasonal Plague (Yersinia pestis) in New Mexico

Plague disease caused by the Gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19th century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen

Detection of <it>Burkholderia pseudomallei</it> O-antigen serotypes in near-neighbor species

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei</it> is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in <it>B</it>. <it>pseudomallei</it>. Complicating this strategy are the four different <it>B. pseudomallei</it> LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of <it>Burkholderia</it> species. Here, we identified the presence of multiple <it>B. pseudomallei</it> O-antigen types and sero-crossreactivity in its near-neighbor species.</p> <p>Results</p> <p>PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of <it>B. mallei</it> and <it>B. thailandensis</it> strains contained the typical O-antigen type A. In contrast, most of <it>B. ubonensis</it> and <it>B. thailandensis</it>-like strains expressed the atypical O-antigen types B and B2, respectively. Most <it>B</it>. <it>oklahomensis</it> strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in <it>B</it>. <it>thailandensis</it> 82172, <it>B</it>. <it>ubonensis</it> MSMB108, and <it>Burkholderia</it> sp. MSMB175. Interestingly, <it>B</it>. <it>thailandensis</it>-like MSMB43 contained a novel serotype B positive O-antigen.</p> <p>Conclusions</p> <p>This study expands the number of species which express <it>B. pseudomallei</it> O-antigen types. Further work is required to elucidate the full structures and how closely these are to the <it>B. pseudomallei</it> O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.</p

Pangenome Analysis of <i>Burkholderia pseudomallei</i>: Genome Evolution Preserves Gene Order despite High Recombination Rates

<div><p>The pangenomic diversity in <i>Burkholderia pseudomallei</i> is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of <i>B</i>. <i>pseudomallei</i> and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.</p></div

Distribution of genes and fit of models described by Haegeman and Weitz [10].

<p>Open circles are data from this study, with fitted lines according to models A (red squares), C (blue filled circles), and D (black triangles). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140274#pone.0140274.t002" target="_blank">Table 2</a> and text for descriptions of models and parameters.</p

Model fit parameters.

<p>Models described by Haegeman and Weitz [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140274#pone.0140274.ref010" target="_blank">10</a>]. Model A: neutral model with all genes exchanged with environment with parameter <i>θ</i>₁. Model C: Genome has a fraction (<i>λ</i>₁) of the genome that is rigid (the core), and the rest exchanges genes with the environment with parameter <i>θ</i>₁. Model D: Similar to model C except the core exchanges genes with the environment with parameter <i>θ</i>₂. The distance from the model fit to the data for <i>B</i>. <i>pseudomallei</i> is given by Δ, with smaller numbers signifying better fit.</p