Temperature and growth rate effects on the hok/sok killer locus for enhanced plasmid stability

The hok/sok locus, isolated from the multiple-resistance plasmid R1 of Escherichia coli, is very efficient at ensuring the stable maintenance of plasmids in Gram-negative systems by killing plasmid-free cells as they arise. To investigate independently the influence of temperature and growth rate on the effectiveness of hok/sok, continuous fermentations have been conducted with the pUC-based, IPTG-induced, beta-galactosidase expression vector pTKW106. At fixed temperature (37 degrees C), decreasing the dilution rate decreased plasmid stability, and at a fixed, low dilution rate (D = 0.15/h), decreasing the temperature resulted in an increase in plasmid stability. These trends are explained by the specific beta-galactosidase activity of each continuous fermentation: higher, specific, recombinant protein expression led to decreased plasmid stability (due to either segregational or structural instability, as determined by plasmid DNA isolation). A representative fed-batch medium produced more beta-galactosidase on a volumetric basis than M9C in the chemostat, and addition of the hok/sok locus increased segregational stability by 8-22-fold in continuous fermentations that lacked antibiotic selection pressure and in which beta-galactosidase was constantly expressed a 12% of total cell protein for 60 h (43-47 generations).