OVERPRODUCTION OF THE ATP-DEPENDENT NUCLEASE ADDAB IMPROVES THE STRUCTURAL STABILITY OF A MODEL PLASMID SYSTEM IN BACILLUS-SUBTILIS

The effect of the ATP-dependent exonuclease AddAB complex on the struc tural stability of plasmid pGP1 in Bacillus subtilis was studied. Usin g deletion mutagenesis and gene amplification techniques, B. subtilis strains were constructed either lacking or overproducing the AddAB com plex, a key enzyme in homologous recombination. The deletion mutant po ssessed no residual ATP-dependent nuclease activity; in contrast, the nuclease activity was up to 30 times higher in lysates of strains carr ying multiple copies of the addAB genes in the chromosome. Southern bl ot analyses of these strains indicated that a linear relationship exis ts between the number of chromosomal gene copies and the level of AddA B activity. The structural stability of pGP1 was analyzed in the AddAB -deficient and over-producing backgrounds. Frequencies of deletion for mation in the plasmid, as monitored by the expression of the pGP1-enco ded penP-lacZ fusion on media containing X-gal, were shown to be incre ased at least 25-fold in the addAB knock-out mutant, whereas the stabi lity of pGP1 was improved up to 15-fold in strains overproducing the A ddAB enzyme. A possible explanation for these findings is that interac tions between AddAB and plasmid molecules prevent the formation of sec ondary structures that constitute potential deletion target sites, and thereby enhance the structural stability of plasmids.