Construction of Plasmids and Bacterial Strains. Salmonella typhi cdtB was amplified from genomic DNA with primers 5'-GGTCATGAAAAAACCTGTTTTTTTCCTTCTG-3' and 5'-CCTCATGACACAGCTTCGTGCCAAAAAGGCTAC-3'. The PCR product was cut with BspHI and cloned into the NcoI site of plasmid pSB616 (1), generating an in-frame carboxyl-terminal fusion with an epitope (M45) derived from the E4-6/7 protein of the adenovirus (2). The DNA segment encoding CdtB M45 was excised with EcoRI and HindIII, and the fragment was made blunt-ended by using the Klenow fragment of DNA polymerase and was subcloned into the EcoRV site of the mammalian expression vector pSB1742 (3). A construct encoding a CdtB derivative lacking its predicted sec-dependent secretion signal sequence was constructed by PCR. Point mutants in the CdtB coding regions resulting in the amino acid change H160Q or D195S were generated by using the QuikChange site-directed mutagenesis system (Stratagene) following the recommendations of the manufacturer. All constructs were verified by nucleotide sequencing. A promoterless luciferase firefly-encoding gene (luc) with a consensus Shine–Dalgarno (AGGA) sequence was amplified by PCR and cloned into the blunt-ended BstEII site of cdtB to generate a transcriptional fusion. This fusion construct was amplified and cloned into the suicide vector pSB890 (4). The cdtB::luc fusion was then mobilized into the S. typhi chromosome by conjugation. The resulting strain was verified by PCR and showed no impairment in its ability to invade cultured Henle-407 intestinal epithelial cells (data not shown).

1. Collazo, C. & Galán, J. E. (1996) Infect. Immun. 64, 3524–3531.

2. Obert, S., O’Connor, R. J., Schmid, S. & Hearing, P. (1994) Mol. Cell. Biol. 14, 1333–1346.

3. Lara-Tejero, M. & Galán, J. E. (2000) Science 290, 354–357.

4. Kaniga, K., Bossio, J. C. & Galán, J. E. (1994) Mol. Microbiol. 13, 555–568.