Localization and inactivation of DNase activity in Clostridium pasteurianum NRRL-B598

Each of four cell fractions of Clostridium pasteurianum NRRL-B598 contained DNase. The culture supernatant contained 31.3% of the total cellular DNase activity, while the cell wash, cell-wall compartmentalized and intracellular fractions contained 27.8, 27.8 and 13.1%, respectively. This enzyme was thermoresistant and heat treatment at various stages of the plasmid isolation protocol reduced chromosomal smearing, but did not improve plasmid recovery. The use of a new density gradient material (cesium trifluoroacetate; CsTFA) known to strongly inactivate DNase was compared to standard isopycnic centrifugation with cesium chloride. The consistent recovery of a 2.3 MDa plasmid only in the presence of CsTFA suggests that DNase continues to be a problem during ultracentrifugation as well as throughout the cleared lysate protocol.