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

Data from: Mutations in global regulators lead to metabolic selection during adaptation to complex environments

Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation

Saxer_et_al_PlosGen_PopulationKey_corrected

This files contains the name of all the populations and ancestors, their selection regime and the accession number for the genomic and proteomics data, if applicable

Saxer_et_al_PlosGen_GrowthOD600_BHI

OD readings of E. coli and C. freundii populations evolved in BHI over the course of a growth cycle in BH

Saxer_et_al_PlosGen_pH

pH of stationary phase cultures of the ancestor and evolved populations of E. coli RU1 and C. freundii RU2 in LB and BHI (in their selective media

Saxer_et_al_PlosGen_CarbohydrateData

Data from carbohydrate analyses of cell extract and spend media from E. coli RU1 ancestor and E. coli RU1-BHI evolved population

Saxer_et_al_PlosGen_GrowthOD600_LB

OD readings of LB-evolved populations of E. coli and C. freundii in LB with the respective ancestor

Mutations in Global Regulators Lead to Metabolic Selection during Adaptation to Complex Environments

<div><p>Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator <i>arcA</i> and the stress response regulator <i>rpoS.</i> We identified 46 unique single-nucleotide variants of <i>arcA</i> and 18 mutations in <i>rpoS</i>, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of <i>rpoS</i> are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting <i>E. coli</i> populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators <i>arcA</i> and <i>rpoS</i> can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation.</p></div

Frequencies of <i>arcA</i> and <i>rpoS</i> mutants in the evolved populations <i>arcA</i> mutations (black bars) reached high frequencies in all LB-evolved populations (A, B) and reach fixation in LB5, while <i>rpoS</i> mutations (red) were more common in BHI-evolved populations (C, D).

<p>The frequencies represent the total frequencies of all <i>arcA</i> or respectively, identified in a particular population.</p