Bacteriophage Mu d1(Ap(r) lac) generates vir-lac operon fusions in Shigella flexneri 2a

Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes, we set out to develop a rapid, easily-assayed phenotype with which to measure expression of virulence. This report described a procedure for isolating vir-lac operon fusions in S. flexneri 2a by using the specialized transducing bacteriophage Mu d1(Ap(r) lac) of Casadaban and Cohen (M. Casadaban and S.N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1976). Mu d1(Ap(r) lac) lysogens were isolated and screened for loss of virulence and for temperature-dependent expression of the lactose genes on Mu d1(Ap(r) lac). A recombinant plasmid carrying the Mu immunity gene was also introduced into lysogens of interest to stabilize the Mu d1(Ap(r) lac) insertion and prevent possible thermal induction at 37°C. The mutant which we isolated failed to penetrate tissue culture cells in the assay for virulence and produced almost 15-fold more β-galactosidase when grown at 37°C than when grown at 30°C. The site of insertion of Mu d1(Ap(r) lac) in this strain was shown to be in the 140-megadalton plasmid pSf2a140, which is known to be associated with virulence. P1L4-mediated transduction of the insertion into a virulent recipient demonstrated genetic linkage of Mu d1(Ap(r) lac) with loss of virulence and temperature-dependent expression of β-galactosidase. All of these features fulfill the phenotype expected for a Mu d1(Ap(r) lac)-induced vir-lac operon fusion. This mutant provides us with a means of measuring expression of a gene function required for virulence by assaying for β-galactosidase. The insertion will also serve as a starting point for mapping of genes on pSf2a140 which are necessary for expression of virulence.