Sedimentation Analysis of Bacterial Nucleoid Structure

The physiology of bacterial DNA topoisomerases can be studied by examining how perturbation of intracellular enzyme activities affects the structure of extracted nucleoids. Since the few DNA nicks that occur when nucleoids are isolated (1 ,2 ) are localized by the presence of 50–100 barriers to strand rotation (2 ,3 ), it is possible to recover chromosomal DNA in which most of each molecule is topologically constrained (2 ,4 ). Consequently, intracellular changes in topoisomerase activity can be detected as differences in the average supercoiling of nucleoids isolated from cells perturbed in different ways. This general strategy has been used to show that supercoiling is relaxed by inhibition of gyrase (5 -7 ) and that it is increased (becomes more negative) by point mutations in topA (the gene encoding topoisomerase I), by low concentrations of gyrase inhibitors, and by anaerobic conditions (6 ,8 -10 ). Experiments of this type have contributed to the conclusions that (1) supercoiling is controlled in part by regulated expression of the gyrase and topoisomerase I genes, and (2) the overall level of supercoiling responds to growth environment (reviewed in [11 ]).