CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene

Abstract

Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene ( galK), we have constructed a plasmid in which the λp re promoter controls galactokinase expression. A galK − host containing this plasmid has a Gal − phenotype since transcription from P RE requires activation by the A CII protein. When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein. A second plasmid was constructed in which the P RE promoter from phage21 controls galactokinase expression. Transcription of the galK gene in this plasmid requires the phage 21 CII protein. Using this system, we demonstrate that the λ and 21 P RE promoters are highly selective for their corresponding CII proteins. However, a cross-reaction between 21 P RE and the λ CII protein was observed. In addition, we transferred the P RE - galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy. Galactokinase expression in this single copy P RE - galK system is also dependent on CII protein, which may be provided from a multicopy plasmid. The high concentration of CII protein provided by the plasmid results in maximal expression of the P RE - galK transcription unit. In this second system low levels of CII activity from CII − mutants are amplified and can be readily detected.