ANALYSIS OF THE STABILIZATION SYSTEM OF PSM19035-DERIVED PLASMID PBT233 IN BACILLUS-SUBTILIS

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed n ine major open reading frames (orfs). The repS, erm1 and erm2 genes ha ve been assigned to three of these orfs, and given the gene order, rep S-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm 1. The organization of genes of the repS-orf gamma region resembles th e organization of genes in the repE-orfI region of pAM beta 1. Messeng er RNA species of molecular weights corresponding to repS, or orf alph a+orf beta, orf gamma, orf delta, and orf epsilon+orf zeta were detect ed by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 k Da correspond to Orfs beta, gamma, delta, epsilon and zeta, respective ly. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were de tected using the T7 promoter-expression system. The orf beta and orf g amma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb seg ment (nt 2999-4552) with coding capacity for orf beta, orf gamma and o rf delta does not seem to affect plasmid maintenance. Removal of a 3.0 -kb fragment (nt 4598-7689) with coding capacity for orf epsilon orfy zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it. On the basis of these observ ations, we conclude that pBT233 stabilization relies on a complex syst em, involving the resolution of plasmid oligomers and additional unkno wn component(s).