Abstract
Gene expression driven by the pR promoter of the λ cI857/pRM/pR system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated pRM promoter, is destabilised at temperatures above 30 °C. In this study, the λ cI857/pRM/pR system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated λ pR 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 λ pR and pRM promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a λ pR 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 pRM activity. The described λ pR 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.
