Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

Bacillus megateriumis gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains ofB. megateriumand closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of thepolyhydroxyalkanoate synthase Cgene ofB. megateriumin designing primers that amplify the 0.9 kb region of thephaCfor its identification. The PCR method was optimized to amplify 0.9 kb region ofphaCgene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only inB. megateriumand was demonstrated by several strains ofB. megateriumisolated from different habitats. The use of Method I did not result in the amplification of thephaCgene with other members of Bacillales. The specificity for identification ofB. megateriumwas confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulatingB. megateriumand members of Bacillales.