Progress is reported in understanding Thiobacillus molecular biology, specifically in the area of vector development. At the initiation of this program, the basic elements needed for performing genetic engineering in T. ferrooxidans were either not yet developed. Improved techniques are described which will make it easier to construct and analyze the genetic structure and metabolism of recombinant T. ferrooxidans. The metabolism of the model organic sulfur compound dibenzothiophene (DBT) by certain heterotrophic bacteria was confirmed and characterized. Techniques were developed to analyze the metabolites of DBT, so that individual 4S pathway metabolites could be distinguished. These techniques are expected to be valuable when engineering organic sulfur metabolism in Thiobacillus. Strain isolation techniques were used to develop pure cultures of T. ferrooxidans seven of which were assessed as potential recombinant hosts. The mixotrophic strain T. coprinus was also characterized for potential use as an electroporation host. A family of related Thiobacillus plasmids was discovered in the seven strains of P. ferrooxidans mentioned above. One of these plasmids, pTFI91, was cloned into a pUC-based plasmid vector, allowing it to propagate in E. coli. A key portion of the cloned plasmid was sequenced. This segment, which is conserved in all of the related plasmids characterized, contains the vegetative origin of DNA replication, and fortuitously, a novel insertion sequence, designated IS3091. The sequence of the DNA origin revealed that these Thiobacillus plasmids represent a unique class of replicons not previously described. The potentially useful insertion sequence IS3091 was identified as a new member of a previously undefined family of insertion sequences which include the E. coli element IS30.