Characterization of an environmental strain of Bacillus thuringiensis from a hot spring in Western Himalayas

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.