Transcription induces a supercoil domain barrier in bacteriophage Mu.

In enteric bacteria, chromosomes are partitioned into domains that exhibit restricted supercoil movement. The most common domain barrier detected by gammadelta resolution assays is random with respect to sequence and occurs more frequently in cells growing rapidly in rich medium compared to cells in stationary phase. Transcription generates both positive and negative supercoiling movement. To address the question of whether transcription causes the appearance of new domain boundaries, a transcriptionally active MudI element was substituted for a MudJr-1 element that resides within the cobT gene of Salmonella typhimurium. Mu-specific transcription from the phage early promoter was placed under control of either the wild type (c(+)) or the temperature-sensitive (cts62) repressor. Using a resolution assay with res sites at six chromosomal locations, domain structure was normal in cells carrying the MudAr-1 prophage with a wild type Mu repressor. However, in cells with a MudAr-1 prophage harboring the cts62 repressor, a new domain barrier appeared in > 90% of the cells. Supercoil movement was restricted ahead of but not behind the transcription machinery. We conclude that the strong Mu early promoter induces the appearance of a domain barrier within the limits of a MudAr-1 prophage.

The DNA cleavage reaction of DNA gyrase. Comparison of stable ternary complexes formed with enoxacin and CcdB protein.

The potent synthetic fluoroquinolones and the natural CcdB protein encoded by the F plasmid both inhibit bacterial growth by attacking DNA gyrase and by stimulating enzyme-induced breaks in bacterial DNA. The cleavage mechanisms of these structurally diverse compounds were analyzed by purifying and characterizing stable ternary complexes of enoxacin and CcdB protein with gyrase bound to a strong gyrase binding site from bacteriophage Mu. Three differences between enoxacin- and CcdB-derived complexes were discovered. 1) Enoxacin binds to the DNA active site and alters the breakage/reunion activity of the enzyme. CcdB binds gyrase-DNA complexes but does not influence enzymatic activity directly. 2) Complexes that produce DNA cleavage with enoxacin are reversible, whereas similar complexes made with CcdB protein are not. 3) Enoxacin stimulates cleavage of both relaxed and supercoiled forms of DNA in the absence of ATP, whereas CcdB induces cleavage only after many cycles of ATP-dependent breakage and reunion. These differences in mechanisms can be explained by a model in which enoxacin induces formation of a novel "cleavable" complex, whereas CcdB protein traps a very rare "cleaved" conformation of the enzyme.