Investigation of the effect of growth environment on the stability of low-copy-number plasmids in Escherichia coli.

The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dmu) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.

Genetical and biochemical aspects of resistance to p-fluorophenylalanine in Saccharomyces cerevisiae.

Growth of haploid yeast strains was inhibited by the phenylalanine (PA) analogue DL-p-fluorophenylalanine (FPA) in yeast extract media containing 0-2 mg PA/ml. Most strains had a maximum FPA tolerance of about 0-25 mg/ml when glycerol was the carbon source and 0-5 mg/ml in in glucose medium. Spontaneous FPA-resistant mutants isolated on glucose medium showed little or no increase in FPA tolerance over that of the parent when metabolizing glycerol. Resistance was controlled by a different nuclear gene in each of four mutants analysed. In a proportion of the mutants the amount of FPA incorporated into cellular proteins in competition with PA was less than into the proteins of sensitive parental cells, whether glucose or glycerol was used as carbon source. This suggests that the mutational change allowed the cytoplasmic system to descriminate against the analogue without affecting its incorporation into mitochondrially-synthesized proteins. Although attempts to measure the latter were not made, the observed decrease in respiratory activity of cells grown in the presence of FPA suggests such incorporation. In other mutants showing resistance to FPA in glucose medium, the amount of FPA incorporated into cellular proteins varied with the carbon source, less analogue being incorporated in glucose medium than in glycerol medium.