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

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

Molecular Basis for the Sorting of the SNARE VAMP7 into Endocytic Clathrin-Coated Vesicles by the ArfGAP Hrb

SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles

Conservation of gene order among pairs of genomes.

<p>Average conservation of gene order for each genome is negatively related to the number of contigs.</p

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

Pairwise values for gene order conservation.

<p>Reds indicate relatively low similarity of gene order and yellows, higher. All values are in the range 0.9089<<i>x</i><0.9980.</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

Pan and core genome size.

<p>The discovery order of genomes was permuted 100 times with the total number of genes discovered shown in upper series, and number of core genes below. The middle series considers genes to be core if they are present in at least <i>n</i>−2 of the genomes (extended core). The bottom series considers them to be core only if present in every strain (strict core). Fitted lines for core homology groups are <i>F</i>_<i>c</i>(<i>n</i>) = <i>κ</i>_<i>c</i> exp[−<i>n</i>/<i>τ</i>_<i>c</i>]+Ω where Ω is the estimated core genome size (dotted lines: 4,568 homology groups for the extended core, and 2,798 for the strict core). Boxes show medians, interquartile range, and full range of data.</p