The properties and molecular structures of two broad host-range streptococcal plasmids are being studied in detail. A 5.0 kbp EcoRI fragment from pAM beta1, shown previously to contain all that is necessary for replication in Streptococcus sanguis, has been cloned in both orientations on pUC19 and on phage M13mp19. A series of nested deletions, in both directions, were obtained by Exonuclease III digestion. The deletions of the M13 clones are currently being used for the nucleotide base sequence analysis of the 5 kbp replicon. The deletions of the pUC19 clones are being cleaved from this vector, blunt-end ligated to streptococcal DNA fragments containing selectable antibiotic resistance determinants, and used in transformation experiments with the Challis strain of S. sanguis. These resistance gene- tagged deletion derivatives are being used to locate regions of this replicon associated with copy number control and incompatibility traits. Similar experiments were also conducted with the 4.2 kbp cryptic plasmid, pVA380-1, which has been used extensively as a cloning vector in streptococcal host-vector systems. Collaborative studies on the use of transposon mutagenesis for genetic and molecular analyses of virulence traits in the Gram-positive pathogens, Bacillus anthracis (with Dr. B. Ivins) and Streptococcus agalactiae (with Dr. S Mattingly), were initiated. Tn916, a conjugative tetracycline resistance transposon from Streptococcus faecalis, was successfully transferred to strains of both species and was also shown to insert at several locations on their respective chromosomes. Auxotrophic mutants of B. anthracis Tn916-containing transconjugants were obtained.
