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Comparative Study
, 108 (12), 5027-32

Bacillus Anthracis Comparative Genome Analysis in Support of the Amerithrax Investigation

Affiliations
Comparative Study

Bacillus Anthracis Comparative Genome Analysis in Support of the Amerithrax Investigation

David A Rasko et al. Proc Natl Acad Sci U S A.

Abstract

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Microbiological identification of morphological variants of B. anthracis Ames. (A) Flowchart used to process the evidentiary material for the identification of the morphological variants. In all cases, the morphotypes are altered in the sporulation phenotype and colony morphology. (B) Image of a representative colony of each of the morphotypes grown on SBA.
Fig. 2.
Fig. 2.
Genomic characterization of morphotype A. (A) No mutation was originally discovered in morphotype A sequencing until a closer examination of the assembly revealed a number of chimeric reads and read pairs that disagreed with the structure of the original assembly, suggesting that a tandem duplication could have occurred. (B) PCR/sequencing assay that was designed to identify other mutations at this locus. The two primers, 1572F and 1572R, are both located within the coding region GBAA_0151, and a PCR amplicon is produced only if the coding region is at least duplicated (Lower). (C) Result of gel electrophoresis of the amplicons for this assay. The genomes amplified are B. anthracis Ames Ancestor (AA), B. anthracis Ames Porton (Porton; not previously known to have this duplication), B. anthracis Ames Leahy letter (LL10), B. anthracis Ames NY Post letter (PL10), and B. anthracis Ames Daschle letter (DL10; triplication of 822-bp amplicon).
Fig. 3.
Fig. 3.
Genomic characterization of morphotypes B, C/D, and E. Each of the loci impacted in these three morphotypes played a role in the sporulation cascade of B. anthracis Ames. Morphotype B (Lower Left) is characterized by an SNP in the promoter region of spoOF, one of the major phosphor-relay proteins in the sporulation cascade. Morphotypes C/D (Upper Left, green) were characterized by either an in-frame deletion (Morph D) or a truncation of the gene product resulting from an SNP in the sensor histidine kinase (GBAA_2291). Morphotype E (Opaque) (Upper Right, blue) is characterized by a 9- or 21-bp deletion in the pXO1 plasmid–encoded Rap phosphatase family protein (GBAA_pXO1_0205).

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