Strain discrimination among B. anthracis and related organisms by characterization of bclA polymorphisms using PCR coupled with agarose gel or microchannel fluidics electrophoresis.
The bclA gene codes for the protein backbone of the exosporium glycoprotein BclA of B. anthracis. BclA has a central collagen-like region formed by polymorphic GXX repeats and conserved amino- and carboxy-termini. It is noted here that the bclA gene is also present in the genome of Bacillus cereus and Bacillus thuringiensis. There is considerable size heterogeneity among the BclA proteins, both for species and strains, due to different numbers of GPT repeats and [GPT]5GDTGTT repeats (BclA repeats). PCR products that included the entire variable region were analyzed by conventional agarose gel electrophoresis and by micro-channel fluidics (MCF) LabChip to assess differences in molecular weight (MW). Both methods provided discrimination at the strain level for B. cereus group organisms. Results obtained by MCF electrophoresis were superior to conventional agarose gel analysis demonstrating improved reproducibility and much faster analysis time. The expression of a carbohydrate-rich exosporium (corresponding to BclA) in other members of the B. cereus group, in addition to B. anthracis, was also demonstrated ultra-structurally. Analysis of sequence variability within the bclA gene CLR revealed even greater potential for strain and species identification.
Department of Pathology and Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208, USA.
Elisangela R
ER
Castanha
Roy R
RR
Swiger
Ben
B
Senior
Alvin
A
Fox
Lashanda N
LN
Waller
Karen F
KF
Fox
D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001409:Bacillus cereus / Q000145:classification* / Q000235:genetics; D015373:Bacterial Typing Techniques; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D046209:Electrophoresis, Microchip / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012430:Ruthenium Red; D013171:Spores, Bacterial / Q000648:ultrastructure
C470593:BclA protein, Bacillus anthracis; D017931:DNA Primers; D008562:Membrane Glycoproteins; D012430:Ruthenium Red