Genomic organization analysis of acidophilic chemolithotrophic bacteria using pulsed field gel electrophoretic techniques

The genomic organization of acidophilic chemolithotrophic bacteria belonging to the genus Thiobacillus, Thiomonas and Leptospirillum was studied using pulsed field gel electrophoresis techniques (PFGE). The electrophoretic analysis of intact DNA prepared from different strains showed that all have a circular chromosome, with sizes ranging from 1.9 Mb for Leptospirillum ferrooxidans ATCC 49879, the smallest genome for an acidophilic strict chemolithoautotrophic microorganism, to 3.8 Mb for Thiomonas cuprina DSM 5495, the largest in this study. The number of extrachromosomal elements present varied from none, as observed in several isolates of Leptospirillum ferrooxidan, to five in Thiobacillus thiooxidans ATCC 8085. The mixotroph Thiomonas cuprina DSM 5495 was found to have a linear 50 kb megaplasmid which was inducible when the bacteria was grown in chemolithotrophic conditions. Low-frequency restriction fragment analysis (LFRFA) of different acidophilic chemolithotrophs and related species was carried out by PFGE to determine macrorestriction patterns for rare cutters (SpeI, XbaI, SwaI, PmeI), which were then used for taxonomic identification (karyotyping), genome size determination, and generation of physical and genetic maps.