9317503 Malamy Conjugal transposons are genetic units that play an important role in the dissemination of genetic information in bacteria. They combine the characteristics of transposons and conjugal elements in a single unit. Conjugal transposons have been extensively studied in the Gram positive cocci: we have recently described Tn4399, a conjugal mobilizing transposon from the important human pathogen, the Gram negative anaerobe Bacteroides fragilis. We are continuing our studies to characterize the transposition and transfer properties of Tn4399. We will concentrate on the proteins and sites within Tn4399 that are required for its ability to transfer from B. fragilis donor cells and to be mobilized by the IncP plasmids, RP4 and R751, in E. coli. We have defined a 2.8 kb region of Tn4399, that contains the "mobilization cassette" which is sufficient for IncP plasmid mobilization. We will determine if this unit can promote plasmid transfer from B. fragilis donor cells, or if other proteins must be supplied in trans. We have identified two Tn4399 proteins, MocA and MocB, and an oriT4399 site that are required for IncP plasmid mobilization in E. coli. We will determine if these proteins, and site are required for Tn4399 promoted transfer in B. fragilis. To develop a genetic tool that can be used to map the B. fragilis chromosome, we will engineer Tm4399 insertions into the B. fragilis chromosome at known locations and determine if B. fragilis chromosomal DNA can now be transferred to recipient cells. To study the transposition properties of Tn4399 we will attempt to establish plasmid-to-plasmid, and chromosome-to plasmid transposition systems. It may be that Tn4399 is not fully competent for transposition and may need proteins supplied in trans from the chromosomal TET transfer element TUF present in the original transposon donor cell. We will test this hypothesis using a new set of plasmids in B.fragilis cells containing TET element TUF on the chromosom e. If these experiments are successful, we will begin to determine the role in transposition of the terminal inverted repeats and the five extra basepairs at the right end of the element, which are a unique feature of Tn4399. The studies of Tn4399 described above should provide important information on the mechanisms of conjugal DNA transfer from B. fragilis donor cells, the mechanism of transposition of this novel elements, and help to create a genetic system for the analysis of the chromosome of B. fragilis. %%% Understanding the interaction of Tn4399 and the IncP broad host range plasmids will provide insights into the dissemination of genetic information to a wide variety of medically important bacterial species. ***
