This study investigated the stability of pHSG415, a low-copy-number plasmid in Escherichia coli strain RV308, under various growth conditions. The plasmid was unstable in batch culture but showed dramatically different stability patterns in continuous culture depending on nutrient limitation. Phosphate, nitrogen, and potassium limitations significantly stabilized the plasmid, while sulphate limitation caused rapid plasmid loss. Increasing dilution rate (growth rate) improved stability under glucose limitation. Analysis using mathematical models revealed that segregational instability—the spontaneous generation of plasmid-free daughter cells at cell division—was the primary cause of plasmid loss across all conditions, not competition from plasmid-free cells with higher growth rates. The parent plasmid pSC101, which contains intact partitioning sequences, remained completely stable under all tested conditions. The findings demonstrate that low-copy-number plasmid stability is highly dependent on growth environment and that nutritional conditions affect segregational stability through mechanisms likely involving the plasmid's partitioning system.

Key findings

• pHSG415 stability varies dramatically with nutrient limitation: stable under phosphate/nitrogen/potassium limitation, but rapidly lost under sulphate limitation in continuous culture
• Segregational instability (spontaneous loss during cell division) is the primary cause of plasmid loss, being 1-2 orders of magnitude greater than any growth rate disadvantage
• Higher dilution rates (faster growth) significantly improve pHSG415 stability, particularly above 0.4 h⁻¹ under glucose limitation
• The deletion of partitioning (par) sequences in pHSG415 compared to parent plasmid pSC101 explains its instability; pSC101 remained 100% stable under all conditions tested
• Growth environment substantially influences plasmid segregational stability, suggesting physiological factors can be manipulated to improve plasmid maintenance in bacterial cultures