Modulation of gene expression by promoter mutants of the lambda cI857/p sub(RM)/p sub(R) system

Gene expression driven by the p sub(R) promoter of the lambda cI857/p sub(RM)/p sub(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p sub(RM) promoter, is destabilised at temperatures above 30 C. In this study, the lambda cI857/p sub(RM)/p sub(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambda p sub(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 C. The impact of the mutations on gene expression levels driven by the lambda p sub(R) and p sub(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambda p sub(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p sub(RM) activity. The described lambda p sub(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.