SUSTAINING PROTEIN-SYNTHESIS IN THE ABSENCE OF RAPID CELL-DIVISION - AN INVESTIGATION OF PLASMID-ENCODED PROTEIN EXPRESSION IN ESCHERICHIA-COLI DURING VERY SLOW GROWTH

The minimum growth rate capable of supporting plasmid-encoded gene expression is determined using continuous cultures of Escherichia coli MZ9387 at dilution rates (D) as low as 5 % of the maximum specific growth rate. Expression from a low copy number plasmid, pMPR166, encoding cyanase under the control of P(lac) is investigated in order to study plasmid-encoded gene expression under conditions approaching starvation. Plasmid copy number was stabilized by selection in the presence of 500 mug/mL chloramphenicol by constitutive expression of chloramphenicol acetyl transferase (CAT). Plasmid retention was determined by dot-blot hybridization and chloramphenicol resistance. The contribution of plasmid maintenance and cyanase expression to the maximum cell yield (Y' x/s) and the maintenance coefficient (m(s)) was determined for MZ9387 and MZ9387:pMPR166 under uninduced and IPTG-induced conditions. The values of Y'x/s and m(s) for non-plasmid-bearing cultures were 0.56 g of cell dry mass (DCM)/g of glucose and 0.26 g of glucose/g of DCM.h, respectively. The cell yield for plasmid-bearing cultures under uninduced conditions (Y-degrees' x/s) was 0.28 g of DCM/g of glucose, with m(s)degrees = 0.08 g of glucose/g of DCM.h. These values decreased following induction of cyanase expression. Glucose consumption in the presence of IPTG was linearly related to the growth rate at D < 0.28 h-1 but nonlinear at dilution rates greater than 50% of the maximum specific growth rate, indicating that cyanase expression alters metabolism and glucose consumption. The fraction of plasmid-free cells decreased with decreasing Damkohler number (Da). These data confirm the usefulness of Da for predicting the relationship between plasmid-free and plasmid-bearing cells where plasmids are stabilized by concentrations of antibiotic greater than the minimum plasmid-free host cell growth inhibitory concentration. Specific cyanase expression increased as the dilution rate decreased to D = 0.15 h-1. Between D = 0.15 h-1 and D = 0.14 h-1, expression decreased 7-fold. At very low dilution rates (D less-than-or-equal-to 0.06 h-1), nonseptated filamentous cells appeared. The appearance of filamentous cells could be reversed by increasing the dilution rate. These data are evidence that when plasmid copy number is stabilized by chloramphenicol resistance, a minimum dilution rate exists below which stringent regulation of protein synthesis dramatically reduces gene expression.