cultivating the bacteria at 37C. Plasmid pCALol (2) was transformed into the E. coli Nissle 1917 cvdA mutant strain to introduce a selective marker To generate an E coli Nissle 1917 cydA napA narG narz mutant, E coil Nissle 1917 cydA pSw172)served as recipient for sequential conjugations and sucrose selections using respectively. Finally, plasmid ps w172 was cured and plasmid pCAL6l introduced by transformation into the E coil Nissle 1917 cydA napA narG narZ mutant strain to introduce a selective marker To generate a cydA mutant of E coli strain MG1655, an internal fragment of the cydA gene was amplified by PCR using the primers MGcydA F GAGGTACCGCATGCGATATCGAGCTATGAATCCGGCTACGAAATG and MGcydA R: TAGCGATCGAATTCCCGGGAGAGCTCCTGGTCGGTAGAACCAGAA. The cydA PCR product was then cloned into suicide vector pGP704 using Gibson Assembly to create pFRCI2. Plasmid pFRC12 was propagated in E coli DHSa apirand introduced into a streptomycin resistant E. coll MG1655 strain by conjugation using E. coliS17-1Apiras a donor strain. Exconjugants were selected on LB agar plates containing streptomycin and carbenicillin to select for integration of the suicide plasmid Chromosomal integration of th plasmid into the cydA gene was verified by PCR and the resulting strain was designated FRC91 Animal Experiments The Institutional Animal Care and Use Committee at the University of California at Davis approved all experiments in this study. Female and male C57BL/6J wild-type mice, aged 8- 10 weeks, were obtained from The Jackson Laboratory. Female and male C57BL/6 Ppargfvillincre-and littermate Pparglvillin /(control )mice were generated at UC Davis by mating Pparg mice with Villin re- mice(The Jackson Laboratory).C57BL/6 Ppargnivillincre-and Pparg Villin /- mice were treated with 20 mg/animal streptomycin via oral gavage or mock treated and orally inoculated 24 hours later with 1 x 10 CFU of 1 mixture of the indicated E. coll or S. Typhimurium strains. For tributyrin supplementation, mice were mock treated or received tributyrin(5 g/kg)by oral gavage at I and 2 days after infection. In some experiments, animals were inoculated with a mixture of 17human infection. When necessary, mice were treated intragastrically at 0, 1, and 2 days after y after Clostridia strains cultures individually in an anaerobic chamber by oral gavage at I da streptomycin with 8 mg/kg/day of the PPARy agonist Rosiglitazone( Cayman) diluted in 0. 1 ml of sterile PBS solution. For the treatment with PPARy antagonist Gw9662( Cayman Ann Arbor, MI), mice were treated intragastrically for 3 days with 5 mg/kg/day of PPARy antagonist GW9662(Cayman, Ann Arbor, Mi)diluted in 0. 2 ml of sterile PBS solution DSS treatment: Mice were given 1% DSS salt(MP Biomedicals)in their drinking water continuously for 8 days as described previously (2). DSS was replaced with fresh DSS solution every 2 to 3 days during this time. At day 4 of DSs treatment, mice were inoculated with 1 x 10% CFU of a 1: I mixture of E coli Nissle 1917 and E. coli Nissle 1917 cydA mutant,or 1: I mixture of a S. Typhimurium invA spiB mutant and a S. Typhimurium invA spiB cya mutant Samples were collected at 4 days after infection Science Author manuscript; available in PMC 2017 October 1cultivating the bacteria at 37°C. Plasmid pCAL61 (2) was transformed into the E. coli Nissle 1917 cydA mutant strain to introduce a selective marker. To generate an E. coli Nissle 1917 cydA napA narG narZ mutant, E. coil Nissle 1917 cydA (pSW172) served as recipient for sequential conjugations and sucrose selections using conjugational donor strains carrying plasmids pSW224, pSW225 and pSW237 (4), respectively. Finally, plasmid pSW172 was cured and plasmid pCAL61 introduced by transformation into the E. coil Nissle 1917 cydA napA narG narZ mutant strain to introduce a selective marker. To generate a cydA mutant of E. coli strain MG1655, an internal fragment of the cydA gene was amplified by PCR using the primers MGcydA_F: GAGGTACCGCATGCGATATCGAGCTATGAATCCGGCTACGAAATG and MGcydA_R: TAGCGATCGAATTCCCGGGAGAGCTCCTGGTCGGTAGAACCAGAA. The cydA PCR product was then cloned into suicide vector pGP704 using Gibson Assembly to create pFRC12. Plasmid pFRC12 was propagated in E. coli DH5α λpir and introduced into a streptomycin resistant E. coli MG1655 strain by conjugation using E. coli S17-1 λpir as a donor strain. Exconjugants were selected on LB agar plates containing streptomycin and carbenicillin to select for integration of the suicide plasmid. Chromosomal integration of the plasmid into the cydA gene was verified by PCR and the resulting strain was designated FRC91. Animal Experiments The Institutional Animal Care and Use Committee at the University of California at Davis approved all experiments in this study. Female and male C57BL/6J wild-type mice, aged 8– 10 weeks, were obtained from The Jackson Laboratory. Female and male C57BL/6 Pparg fl/flVillin cre/− and littermate Pparg fl/flVillin −/− (control) mice were generated at UC Davis by mating Pparg fl/fl mice with Villin cre/− mice (The Jackson Laboratory). C57BL/6, Pparg fl/flVillin cre/− and Pparg fl/flVillin −/− mice were treated with 20 mg/animal streptomycin via oral gavage or mock treated and orally inoculated 24 hours later with 1 × 109 CFU of 1:1 mixture of the indicated E. coli or S. Typhimurium strains. For tributyrin supplementation, mice were mock treated or received tributyrin (5 g/kg) by oral gavage at 1 and 2 days after infection. In some experiments, animals were inoculated with a mixture of 17 human Clostridia strains cultures individually in an anaerobic chamber by oral gavage at 1 day after infection. When necessary, mice were treated intragastrically at 0, 1, and 2 days after streptomycin with 8 mg/kg/day of the PPARγ agonist Rosiglitazone (Cayman) diluted in 0.1 ml of sterile PBS solution. For the treatment with PPARγ antagonist GW9662 (Cayman, Ann Arbor, MI), mice were treated intragastrically for 3 days with 5 mg/kg/day of PPARγ antagonist GW9662 (Cayman, Ann Arbor, MI) diluted in 0.2 ml of sterile PBS solution. DSS treatment: Mice were given 1% DSS salt (MP Biomedicals) in their drinking water continuously for 8 days as described previously (2). DSS was replaced with fresh DSS solution every 2 to 3 days during this time. At day 4 of DSS treatment, mice were inoculated with 1 × 109 CFU of a 1:1 mixture of E. coli Nissle 1917 and E. coli Nissle 1917 cydA mutant, or 1:1 mixture of a S. Typhimurium invA spiB mutant and a S. Typhimurium invA spiB cyxA mutant. Samples were collected at 4 days after infection. Byndloss et al. Page 8 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript