| I have studied the enzymes and cellular structures that control chromosome topology in Escherichia coli. These are: DNA gyrase and topoisomerase (topo) IV, the two type II topoisomerases in E. coli; and supercoiling domain barriers, which divide the bacterial chromosome into topologically independent units. First, I investigated the mode of action of the gyrase inhibitor, novobiocin, on topo IV. I found that topo IV is a secondary target of novobiocin in vitro and in vivo, and that inhibition of topo IV occurs by the same mechanism as that for gyrase. I also established that unlike the pathway leading to high-level quinolone resistance in clinical E. coli isolates, sequential mutation of gyrase and topo IV to resistant alleles does not confer an additive increase in novobiocin resistance.; Next, I studied the structure of whole E. coli chromosomes by electron microscopy. This work showed that the bacterial DNA is organized into individual loops and that some loops could be relaxed while the rest of the chromosome remained supercoiled. The supercoiled loops I measured were highly variable in length and were much smaller than previous estimates for topological domain sizes. A subsequent study from our lab showed that domains measured in vivo are, in fact, five to ten-fold smaller than previously reported and that their sizes are stochastic. Thus, the in vivo and in vitro data match in both the average length of domains, 10 kb, and the random distribution of their sizes.; Finally, in an effort to define the cellular components responsible for forming topological domain barriers on the chromosome, I designed a genetic selection for supercoiling-deficient mutants of E. coli. Using this novel approach, I identified five genes, along with gyrA and gyrB, whose disruption reduced chromosomal supercoiling density in vivo. These are: hns, dksA, fis, pgm and tktA. Deletion of any of these genes resulted in sensitivity to novobiocin and to DNA damage, suggesting that their products, or those encoded co-cistronically, are involved in maintaining topological structure of the E. coli chromosome. |
