Small colony, dwarf or variant strains of bacteria which are isolated following exposure of sensitive populations to the aminoglycoside antibiotic gentamycin, are low-level-resistant anaerobic mutants with defective electron transport and membrane- localized ATP synthesis. Thus, they are blocked in the transport of this class of antibiotics. These strains are the result of a pleiotropic mutation in a gene which controls one component of the electron transport system. The long term objective of this research project is to determine the role that transposable elements play in the induction of these strains in Staphylococcus aureus and E. coli. It is believed that IS elements or transposons are involved in the formation of these strains because: 1) in S. aureus plasmid loss or modification accompanies the formation of the variants; 2) variants showing plasmid loss (most likely not due to decrease in copy number) can be induced to revert, with the appearance of new and original plasmids; 3) the induction of the variant strains appears to result from an induced process, and 4) in E. coli more than 80% of the variants carry mutations in the hem B gene with the remaining 20% falling into the hem A and hem C genes. The immediate goals or specific aims and the methods to be used are as follows: 1) the establishment of comparative physical maps of the S aureus parent plasmids and variant and revertant plasmids which show an increase or decrease in molecular weight. This will be done by restriction endonuclease analysis; 2) Southern hybridization using probes of known IS elements will be carried out to identify the presence of transposable elements in the modified plasmids of variant and revertant strains of S. aureus; 3) direct cloning of the mutant hem B gene from existing variant strains of E. coli, and the analysis of these genes by restriction analysis and Southern hybridization for evidence of the insertion of transposable elements; 4) confirmation of evidence for transposable element activity by electron microscopy heteroduplex mapping, and 5) further development of the parameters of a novel system in S. aureus in which reversion of the slow growing variant strains can be induced by the presence of antibiotics such as tetracycline.
