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 nine major open reading frames ( orfs ). The repS, erm1 and erm2 genes have been assigned to three of these orfs , and given the gene order, repS- orfα- orfβ- orfγ- orfδ- orfϵ- orfζ-erm2-erm1. The organization of genes of the repS- orfy region resembles the organization of genes in the repE- orfI region of pAMβ1. Messenger RNA species of molecular weights corresponding to repS, orfα + orf β, orfγ, orfδ and orfϵ + orfζ were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs β, γ, δ, ϵ and ζ, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orfβ and orfγ encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs , δ, ϵ and ζ, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999–4552) with coding capacity for orfβ, orfγ and orfδ does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598–7689) with coding capacity for orfϵ and orfζ reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546–7826) abolished it. On the basis of these observations, we conclude that pBT233 stabilization relies on a complex system, involving the resolution of plasmid oligomers and additional unknown component (s).