Stability of a recombinant shuttle plasmid in Bacillus subtilis and Escherichia coli

The recombinant shuttle plasmid pCPPS-31, which confers carboxymethylcellulase (CMCase) production and neomycin resistance (Ner), was segregationally but not structurally unstable in both B. subtilis (50-60%) and Escherichia coli (80-90%) when grown in serial batch cultures for 72 h diluting 1:1,000 with fresh medium every 24 h. The plasmid showed 80-100% segregational and complete structural stability in B. subtilis during batch, fed-batch, and continuous culture fermentations in minimal M9 medium. In E. coli the instability was > or = 50% in 36 h of batch or continuous culture and approached 100% in 72 h of continuous fermentation. The plasmid was more stable (> or = 60%) during fed-batch fermentation. Stability in B. subtilis and instability in E. coli may be attributed to (i) the significant growth-rate advantage of plasmid-free (P-) cells over plasmid-harboring (P+) cells in the case of E. coli, an advantage that was absent in B. subtilis, and (ii) the existence of plasmid in oligomeric forms in E. coli and in mainly monomeric forms in B. subtilis.