This research proposal deals with a novel class of transposons capable of intracellular as well as intercellular transposition. Besides being causative agents for the horizontal spread of multiple antibiotic resistance among clinical streptococci, this group of genetic elements has been shown to transfer from Gram positive to Gram negative bacteria. Thus, an investigation of the properties of these is of biological interest, and of medical relevance. Experimental observations seem to indicate that the larger conjugative transposons such as omegaBM6001 and omegaBM4200 are made of smaller discrete units at least one of which is capable of independent movement when removed from its original context. The objectives of the proposed study are to distinguish between and to isolate the component units, and to identify the transfer related regions. Recent experiments demonstrating the retention of conjugal properties of the BM6001 element subsequent to the in vivo deletion of a Tn916 like element from within it strongly suggest the existence of a separate class class of mobile elements whose properties could be distinct from those of the Tn916 class of elements. Mutant donor strains would be created to define the transfer-- related regions within the omegaBM6001 devoid of the tet element. The details of the preferred target sites for the omegaBM6001 and the omegaBM4200 elements would be obtained by dideoxy DNA sequencing. The role of the termini and the preferred target site of the BM6001 element in conjugal transfer would be tested following construction of mutant strains carrying in vivo induced deletions of these regions and using them in filter-mating experiments. Besides characterizing the structural and functional details of this novel class of transposable DNA segments, the proposed investigation could enlarge our understanding of the origin and autoaccumulation of heterologous elements some of which include antibiotic resistance determinants.
