SURVEYING A SUPERCOIL DOMAIN BY USING THE GAMMA-DELTA RESOLUTION SYSTEM IN SALMONELLA-TYPHIMURIUM

A genetic system was developed to investigate the supercoil structure of bacterial chromosomes. New res-carrying transposons were derived fr om MudI1734 (MudJr1 and MudJr2) and Tn10 (Tn10dGn). The MudJr1 and Mud Jr2 elements each have a res site in opposite orientation so that when paired with a Tn10dGn element in the same chromosome, one MudJr res s ite will be ordered as a direct repeat. Deletion formation was studied in a nonessential region (similar to 100 kb) that extends from the hi s operon through the cob operon. Strains with a MudJr insertion in the cobT gene at the 5' end of the cob operon plus a Tn10dGn insertion po sitioned either clockwise or counterclockwise from cobT were exposed t o a burst of RES protein. Following a pulse of resolvase expression, d eletion formation was monitored by scoring the loss of the Lac(+) phen otype or by loss of tetracycline resistance. In exponentially growing populations, deletion products appeared quickly in some cells (in 10 m in) but also occurred more than an hour after RES induction. The frequ ency of deletion (y) diminished with increasing distance (x) between r es sites. Results from 15 deletion intervals fit the exponential equat ion y = 120 . 10((-0.02x)). We found that res sites can be plectonemic ally interwound over long distances (>100 kb) and that barriers to sup ercoil diffusion are placed stochastically within the 43- to 45-min re gion of the chromosome.