title: Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.
abstract: BACKGROUND
  Despite 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).
  
  
  RESULTS
  Archival 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: BioSciences Division, Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, United States of America. henry.gibbons@us.army.mil
authors.forename: Henry S
authors.initials: HS
authors.lastname: Gibbons
authors.forename: Stacey M
authors.initials: SM
authors.lastname: Broomall
authors.forename: Lauren A
authors.initials: LA
authors.lastname: McNew
authors.forename: Hajnalka
authors.initials: H
authors.lastname: Daligault
authors.forename: Carol
authors.initials: C
authors.lastname: Chapman
authors.forename: David
authors.initials: D
authors.lastname: Bruce
authors.forename: Mark
authors.initials: M
authors.lastname: Karavis
authors.forename: Michael
authors.initials: M
authors.lastname: Krepps
authors.forename: Paul A
authors.initials: PA
authors.lastname: McGregor
authors.forename: Charles
authors.initials: C
authors.lastname: Hong
authors.forename: Kyong H
authors.initials: KH
authors.lastname: Park
authors.forename: Arya
authors.initials: A
authors.lastname: Akmal
authors.forename: Andrew
authors.initials: A
authors.lastname: Feldman
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Lin
authors.forename: Wenling E
authors.initials: WE
authors.lastname: Chang
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Higgs
authors.forename: Plamen
authors.initials: P
authors.lastname: Demirev
authors.forename: John
authors.initials: J
authors.lastname: Lindquist
authors.forename: Alvin
authors.initials: A
authors.lastname: Liem
authors.forename: Ed
authors.initials: E
authors.lastname: Fochler
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Read
authors.forename: Roxanne
authors.initials: R
authors.lastname: Tapia
authors.forename: Shannon
authors.initials: S
authors.lastname: Johnson
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Bishop-Lilly
authors.forename: Chris
authors.initials: C
authors.lastname: Detter
authors.forename: Cliff
authors.initials: C
authors.lastname: Han
authors.forename: Shanmuga
authors.initials: S
authors.lastname: Sozhamannan
authors.forename: C Nicole
authors.initials: CN
authors.lastname: Rosenzweig
authors.forename: Evan W
authors.initials: EW
authors.lastname: Skowronski
mesh_terms: D000483:Alleles; D001407:Bacillus / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D020029:Base Pairing / Q000235:genetics; D054045:Biological Warfare Agents; D002374:Catalase / Q000378:metabolism; D015169:Colony Count, Microbial; D019295:Computational Biology; D004252:DNA Mutational Analysis; D019143:Evolution, Molecular; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D005838:Genotype; D054643:INDEL Mutation / Q000235:genetics; D055442:Metabolome / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013171:Spores, Bacterial / Q000235:genetics
chemical_list: D054045:Biological Warfare Agents; D002374:Catalase