lacZ fusions to genes that specify exported proteins: a general technique

We have devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for beta-galactosidase (lacZ). These fusions specify a hybrid protein comprised of an NH2-terminal portion of the exported protein and a large functional COOH-terminal portion of beta-galactosidase. The fusions are constructed with a derivative of the MudII (lac, Ap) phage. To overcome the lethality that is often associated with the expression of such a hybrid gene, we have recombined an early lacZ nonsense mutation onto this phage. With the use of strains that carry a temperature-sensitive nonsense suppressor, expression of the full-length hybrid protein can be controlled by varying the growth temperature. We demonstrated the utility of this technique by isolating a series of fusions to a gene, ompA, coding for a major outer membrane protein. As expected, strains containing these fusions are not viable under conditions that permit synthesis of a functional nonsense suppressor. Accordingly, this method should also be useful for direct selection of export-defective mutants.