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

Pathoadaptive Mutations in Salmonella enterica Isolated after Serial Passage in Mice

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth

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

Expression levels of <i>spvA</i> (black bars), <i>mgtA</i> (light grey bars) and <i>rpoS</i> (dark grey bars) in the StpA(E42Del) and the PhoQ(L13Q) mutant relative to wild type levels.

<p>All expression levels are relative to the <i>dnaE</i> mRNA levels in each RNA sample. Error-bars are SEM.</p

Strains of <i>S. typhimurium</i> used in this study.

<p>Strains of <i>S. typhimurium</i> used in this study.</p

Competitive indexes of reconstructed and parental mutants.

<p>Competitive indexes of reconstructed and parental mutants.</p

Motility testing on motility agar.

<p>A) Illustrating how motility is scored on motility agar. Motility is scored on a 5 step scale where (−) is non-motile and+++is fully motile. B) Motility and phase variation of wild type and evolved Salmonella typhimurium strains. FljBA^ON cells expressing <i>fljB</i> type of flagellum and FljBA^OFF cells expressing <i>fliC</i> type of flagellum. Percentage indicates the fraction of cells (based on whole-genome sequencing) in mouse-adapted population that were in FljBA^ON state.</p

IS Element Variation Between Strains.

<p>*<i>pldA/IS100</i> chimeric reads are present in a subset of 25% of reads that map to this position (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604.s002" target="_blank">Figure S2</a>). The remainder of the reads corresponded to an intact <i>pldA</i> gene.</p

Genetic Diversity of the 2009 Plague Isolates.

<p>Distribution of mutations through the 2009 isolates. Mutations relative to the parent strain (CO92) are indicated by black squares. Grey squares indicate that the mutation was not called automatically but was evident by manual inspection of the assembly. Mutation 31 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone-0031604-t002" target="_blank">Table 2</a> is not shown nor incorporated into the phylogeny as it is an expansion of a 10 bp repeating sequence.</p