Construction of mutant strains and plasmids

The bacterial plasmids and primers used are listed in S2 and S3 Tables, respectively. For the construction of S. Typhimurium TH1624 (T1), an invA::pEP185.2 [23] was transduced from IR715 into SH100 using a P22 phage. Gene deletions of S. Typhimurium were constructed by the PCR-based λ Red recombination system [24]. For construction of the double-gene deletion in S. Typhimurium, TH1821 (ΔgogAΔpipA::Km), a ΔpipA::Km mutation from strain TH1772, was introduced by P22 phage, after elimination of the chloramphenicol (Cm) cassette from strain TH1671 (ΔgogA::Cm) using pCP20. Using similar methods, other double-deletion strains (TH1681 [ΔgogAΔgtgA] and TH1820 [ΔgtgAΔpipA::Km]) and multi-deletion mutants (TH1779 [ΔgogAΔgtgAΔpipA::Km], TH1722 [ΔgogAΔgtgAΔpipAΔsseK1ΔsseK2ΔsseK3::KmΔsteE::Cm], TH1586 [ΔsseK1ΔsseK2ΔsseK3::Km], TH1766 [invA::pEP185.2 ΔgogAΔgtgA], and TH1770 [ssaV::Cm ΔgogAΔgtgA]) were constructed. All mutations were verified by PCR.

gogA, gtgA, and pipA were amplified by PCR and cloned into pTAKN-2 (BioDynamics Laboratory, Tokyo). The PCR product for gtgA was digested with BamHI to distinguish between gogA and gtgA. After TA cloning, these genes were subcloned into a plasmid pFLAG-CTC (Sigma, St. Louis, MO) for the expression of FLAG-fusion proteins in E. coli and S. Typhimurium, a plasmid pMW118 (Nippon Gene, Tokyo, Japan) for a complementation assay or a plasmid pEGFP-C1 (Clontech, Mountain View, CA) for expression of EGFP-fusion proteins in mammalian cell lines. Point mutations (on GogA, GtgA, or PipA) were constructed with overlapping primers, TH816 and TH817, TH765 and TH766, or TH846 and TH847, respectively, using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). These mutations were verified by DNA sequencing.

pEGFP-NleC [25] and pCMV-FLAG-p65 were provided by Drs. Toru Tobe (Osaka University) and Hiroshi Ashida (Tokyo Medical and Dental University), respectively.