Bindloss et al the microbial community towards a dominance of obligate anaerobes, which produce short chain fatty acids. In turn, short-chain fatty acids sustain Tregs and epithelial PPAR-y signaling, which cooperatively drives the energy metabolism of colonocytes towards B- oxidation of microbiota-derived butyrate to preserve epithelial hypoxia, thereby closing a virtuous cycle maintaining homeostasis of a healthy gut. PPAR-y-signaling also activates expression of beta-defensins, which might contribute to shaping the intestinal environment (25). An antibiotic-induced lack of epithelial PPAR-y-signaling and a contraction of the treg pool cooperatively drive a metabolic reorientation of colonocytes towards anaerobic glycolysis, thereby increasing epithelial oxygenation and consequently elevating oxygen bioavailability to promote an expansion of Enterobacteriaceae(Fig. SD), a common marker of dysbiosis(1). Thus, an expansion of Enterobacteriaceae in the gut-associated microbial community is a microbial signature of epithelial dysfunction, which has important ramifications for targeting PPAR-y-signaling as a potential intervention strategy MATERIALS AND METHODS Bacterial culture conditions The 17 human Clostridia isolates were kindly provided by K. Honda(20, 26)and were cultured individually as described previously (10). E. coli strains( wild-type E coli Nissle 1917, E. coli Nissle 1917 napA narZ narG mutant (2), E. coli Nissle 1917 cydAB mutant, E. coll Nissle 1917 cydAB napA narZ narG mutant, E colI JB2 (27), E colI MG1655, E. coll MG1655 cydA mutant)and S. Typhmurium strains(wild-type ATCC14028 resistant to nalidixic acid []. Ir715 invA spiB mutant, IR715 cyxA mutant or IR715 InvA spiB cyxA mutant)(3)were routinely grown aerobically at 37 C in LB broth(BD Biosciences)or on LB plates. When necessary, antibiotics were added to the media at the following concentrations: 0. I mg/ml carbenicillin, and 0.05 mg/ml kanamycin Single colonies of endogenous coliforms isolated on McConkey agar from the feces of Charles river mice were subjected to species identification using EnteroPluri Test (Liofilchem, Italy ) per the manufacturer's recommendations Construction of e. coli mutants To construct an E coli Nissle 1917 cydA mutant, upstream and downstream regions of approximately 0.5kb in length flanking the cyda gene were amplified by PCr and purified using the MiniElute kit( Qiagen). The pRDH1O suicide vector was digested with Sall, run on an agarose gel, purified using the MiniElute kit(Qiagen) and assembled with the fragments using the Gibson Assembly Master Mix(NEB)to form plasmid pYl9 Plasmid pYL9 was nd conjugation performed with E. coli Nissle 1917 carrying the temperature-sensitive plasmid pSw172 for counter selection(4). Conjugation was performed at 30oC and exconjugants in which the suicide plasmid had integrated into the chromosome were recovered on LB plates containing carbenicilin and chloramphenicol Subsequent sucrose selection was performed on sucrose plates(5% sucrose,& g/l nutrient broth base, 15 g/l agar)to select for a second crossover events. PCR was performed to detect events that lead to the unmarked deletion of the cydA gene Plasmid pswI72 was cured by Science Author manuscript; available in PMC 2017 October 1The emerging picture is that epithelial hypoxia maintains anaerobiosis in the colon to drive the microbial community towards a dominance of obligate anaerobes, which produce short￾chain fatty acids. In turn, short-chain fatty acids sustain Tregs and epithelial PPAR-γ- signaling, which cooperatively drives the energy metabolism of colonocytes towards β- oxidation of microbiota-derived butyrate to preserve epithelial hypoxia, thereby closing a virtuous cycle maintaining homeostasis of a healthy gut. PPAR-γ-signaling also activates expression of beta-defensins, which might contribute to shaping the intestinal environment (25). An antibiotic-induced lack of epithelial PPAR-γ-signaling and a contraction of the Treg pool cooperatively drive a metabolic reorientation of colonocytes towards anaerobic glycolysis, thereby increasing epithelial oxygenation and consequently elevating oxygen bioavailability to promote an expansion of Enterobacteriaceae (Fig. S1), a common marker of dysbiosis (1). Thus, an expansion of Enterobacteriaceae in the gut-associated microbial community is a microbial signature of epithelial dysfunction, which has important ramifications for targeting PPAR-γ-signaling as a potential intervention strategy. MATERIALS AND METHODS Bacterial culture conditions The 17 human Clostridia isolates were kindly provided by K. Honda (20, 26) and were cultured individually as described previously (10). E. coli strains (wild-type E. coli Nissle 1917, E. coli Nissle 1917 napA narZ narG mutant (2), E. coli Nissle 1917 cydAB mutant, E. coli Nissle 1917 cydAB napA narZ narG mutant, E. coli JB2 (27), E. coli MG1655, E. coli MG1655 cydA mutant) and S. Typhmurium strains (wild-type ATCC14028 resistant to nalidixic acid [IR715], IR715 invA spiB mutant, IR715 cyxA mutant or IR715 invA spiB cyxA mutant) (3) were routinely grown aerobically at 37°C in LB broth (BD Biosciences) or on LB plates. When necessary, antibiotics were added to the media at the following concentrations: 0.1 mg/ml carbenicillin, and 0.05 mg/ml kanamycin. Single colonies of endogenous coliforms isolated on McConkey agar from the feces of Charles River mice were subjected to species identification using EnteroPluri Test (Liofilchem, Italy) per the manufacturer`s recommendations. Construction of E. coli mutants To construct an E. coli Nissle 1917 cydA mutant, upstream and downstream regions of approximately 0.5kb in length flanking the cydA gene were amplified by PCR and purified using the MiniElute kit (Qiagen). The pRDH10 suicide vector was digested with SalI, run on an agarose gel, purified using the MiniElute kit (Qiagen) and assembled with the fragments using the Gibson Assembly Master Mix (NEB) to form plasmid pYL9. Plasmid pYL9 was then transformed into E. coli S17-1 λpir and conjugation performed with E. coli Nissle 1917 carrying the temperature-sensitive plasmid pSW172 for counter selection (4). Conjugation was performed at 30°C and exconjugants in which the suicide plasmid had integrated into the chromosome were recovered on LB plates containing carbenicilin and chloramphenicol. Subsequent sucrose selection was performed on sucrose plates (5 % sucrose, 8 g/l nutrient broth base, 15 g/l agar) to select for a second crossover events. PCR was performed to detect events that lead to the unmarked deletion of the cydA gene. Plasmid pSW172 was cured by Byndloss et al. Page 7 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript