Bradyrhizobium sp. strain 32H1 cells express a number of bacteroid-associated functions and repress some functions related to the free-living state when grown ex planta under conditions of low (0.2%) oxygen tension and relatively high levels (>8 mM) of medium K. Expression of the bacteroid-associated phenotype was blocked by the DNA gyrase inhibitor novobiocin. Because the degree of negative supercoiling of DNA is the result of the activities of both DNA gyrase and topoisomerase I, we measured these enzymes in cells grown under nitrogen-fixing (low O(2), high K) and non-nitrogen-fixing conditions (low O(2), low [50 muM] K or high O(2), high K). Lower topoisomerase I activities were seen in extracts from nitrogen-fixing cells than in those from non-nitrogen-fixing cells. In contrast, DNA gyrase levels were lower in high-O(2)-grown cells than under the other conditions tested. These differences are consistent with an increase in DNA superhelicity associated with growth under low-O(2), high-K conditions. A spontaneous mutant resistant to the DNA gyrase inhibitor ciprofloxacin was found to be constitutive with respect to the K requirement, because it expressed the bacteroid-associated phenotype when grown under low-O(2), low-K conditions. The mutant cells gave rise to effective nodules on Macroptilium atropurpureum and possessed the low topoisomerase I activities and high DNA gyrase levels of low-O(2)-, high-K-grown wild-type cells. Our data suggest that changes in DNA supercoiling resulting from low O(2) tension and a high K concentration exert a major influence on the expression of the bacteroid-associated phenotype.