Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

Henry S Gibbons,Hajnalka Daligault,Chris Detter,Cliff Han,Lauren A Mcnew,Michael Krepps,Jeffrey S Lin,C Nicole Rosenzweig,Evan W Skowronski,Brandon W Higgs,Kyong H Park,Timothy D Read,Ed Fochler,Shanmuga Sozhamannan,Wenling E Chang,Carol Chapman,David Bruce,Arya Akmal,Roxanne Tapia,S M Broomall ,Charles C Hong ,John L Lindquist ,Andrew B Feldman ,Plamen A Demirev ,Kimberly A Bishop‐Lilly ,Alvin T Liem ,Shannon L Johnson ,Mark A Karavis ,Paul Mcgregor

doi:10.1371/journal.pone.0017836

Abstract

BackgroundDespite the decades-long use of Bacillus atrophaeus var. globigii (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).ResultsArchival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis 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.ConclusionsOur 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.

Highlights

Bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to B. subtilis
Other than the formation of the dark pigment, it is virtually indistinguishable from B. subtilis by conventional phenotypic analysis [13], and the lack of distinguishing metabolic or phenotypic features has contributed to the confusionin the taxonomic placement of this organism
We report the definitive molecular typing of several BGstrains using whole-genome sequences, and develop a plausible microevolutionary history of a commonly used lineage based on the accumulation of mutations over time and during transfer between laboratories.The selected strains span more than six decades of development, use, and transfer of BGbetween various institutions and laboratories and offer an unparalleled opportunity to investigate mutation under selection and drift over time

Summary

Introduction

Bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to B. subtilis. Isolated as B. globigii in 1900 (Migula) as a variant of B. subtilis, it was originally distinguished from B. subtilis by the formation of a black-tinted pigment on nutrient agar and by low rates of heterologous gene transfer from B. subtilis [11]. It has been alternately known as B. subtilis var. Other than the formation of the dark pigment, it is virtually indistinguishable from B. subtilis by conventional phenotypic analysis [13], and the lack of distinguishing metabolic or phenotypic features has contributed to the confusionin the taxonomic placement of this organism. 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)

Methods

Results

Conclusion