Identification of a region of genetic variability among Bacillus anthracis strains and related species

Abstract

The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR ''fingerprints'' generated by an M13 bacteriophagederived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine-and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.

Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats

“…Other examples of bacterial VNTR loci occur inside ORFs (Andersen et al, 1996;Frknay et al, 1994). As expected all reported VNTR loci within bacterial coding regions have repeat sizes which are multiples of three.…”

“…VNTR loci are also used for human forensic and paternity testing. Individual VNTR loci have been identified in bacteria (Andersen et al, 1996;Frknay et al, 1994;Frothingham, 1995;Goyal et al, 1994) but no systematic analysis has been reported. We undertook a systematic analysis of the variability of tandem repeat loci in the M .…”

Forensic microbiology and bioterrorism risk (Part II)

“…Il B. anthracis possiede un DNA cromosomico e due plasmidi, pOX1 38 e pOX2 39 , sui quali si trovano i geni codificanti per i fattori di virulenza: la capsula 40 nel primo e la tossina dell'antrace 41 nel secondo, la quale è costituita da 3 subunità, letal factor (LF), edema factor (EF) e protective antigen (PA), ognuna codificata da un gene diverso 42 . Proprio sul B. anthracis furono sviluppati numerosi studi con lo scopo di individuarne i polimorfismi fino a quando, tra il 1996 e il 1997, due gruppi di scienziati arrivarono all'identificazione di 8 loci genici contenenti sequenze VNTR, che mostrarono un elevato grado di variabilità fra i diversi ceppi analizzati 43,44 . Per l'identificazione di questi loci furono sequenziati diversi frammenti di DNA attraverso la tecnica amplified fragment length polymorphism (AFLP) 45 , utilizzata anche per la determinazione delle relazioni filogenetiche del bacillo dell'antrace con gli altri bacilli della stessa famiglia (B. cereus e B. thuringiensis) 43,44,46 .…”

“…Proprio sul B. anthracis furono sviluppati numerosi studi con lo scopo di individuarne i polimorfismi fino a quando, tra il 1996 e il 1997, due gruppi di scienziati arrivarono all'identificazione di 8 loci genici contenenti sequenze VNTR, che mostrarono un elevato grado di variabilità fra i diversi ceppi analizzati 43,44 . Per l'identificazione di questi loci furono sequenziati diversi frammenti di DNA attraverso la tecnica amplified fragment length polymorphism (AFLP) 45 , utilizzata anche per la determinazione delle relazioni filogenetiche del bacillo dell'antrace con gli altri bacilli della stessa famiglia (B. cereus e B. thuringiensis) 43,44,46 . In particolare, 6 degli 8 loci polimorfici furono individuati sul DNA cromosomiale (CG3,vrrA,vrrB1,vrrB2,vrrC1,vrrC2) e altri due (pXO2-at e pXO1-aat) sui due plasmidi 43,44 .…”

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…[13][14][15] The first DNA signatures that were developed for anthrax PCR detection methods independently of plasmids occurrence were DNA fragments used to genotype B. anthracis. They include the vrrA marker, [26][27][28] the AC-390 gene, 29 and the SG-850/749 fragment. 30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps.…”