The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The site-specific resolution systems of closely related transposons Tn3 and γδ are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000. © 2005 Blackwell Publishing Ltd.