Epitope tagging of chromosomal genes inSalmonella

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000)Proc. Natl. Acad. Sci. USA97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and
includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion
of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage ʎredfunctions
yields recombinants carrying the targeted gene fused to the
epitope-encoding sequence. The resulting C-terminal-tagged protein
can be identified by standard immuno-detection techniques. In
an initial application of the method, we have added the sequences
encoding the FLAG and 3xFLAG and influenza virus hemagglutinin
epitopes to various genes ofSalmonella entericaserovar Typhimurium,
including putative and established pathogenic determinants
present in prophage genomes. Epitope fusion proteins were
detected in bacteria growingin vitro, tissue culture cells, and
infected mouse tissues. This work identified a prophage locus
specifically expressed in bacteria growing intracellularly. The procedure
described here should be applicable to a wide variety of
Gram-negative bacteria and is particularly suited for the study of
intracellular pathogens.