Immediately after the pulse, the cells were cooled on ice for 3 min, suspended in 1 mL of TSBHKFN for 24 h at 37 °C for recovery, and then streaked on a blood agar plate containing 1 μg mL−1 erythromycin. Plates were kept under standard anaerobic conditions for 4 weeks. Erythromycin-resistant colonies were subjected to Southern blot and reverse transcription (RT)-PCR analyses for verification of the insertional inactivation of the TF0022 locus by double cross-over recombination (Fig. S1). Total RNA was extracted from T. forsythia cells with the RiboPure-Bacteria kit (Ambion, Austin, TX), according to the manufacturer’s instructions.

U0126 mouse Semi-quantitative RT-PCR was performed using SuperScript III (Invitrogen) and random primers for cDNA synthesis followed by 22–25 cycles of PCR with 400 ng of template cDNA, gene-specific primers (TF0022-Fw and TF0022-Rv as listed in Table S1), and KOD-plus DNA polymerase (Toyobo, Osaka, Japan). Cultures Anti-diabetic Compound Library order of wild-type T. forsythia and the TF0022-ko mutant were normalized in 5 mL of TSBHKFN in culture tubes to an OD600 nm

of approximately 0.5 and left to stand at 37 °C under anaerobic conditions. Samples (100 μL) were taken 1 cm below the surface of the culture at the beginning of the assay and after 2, 4, 6, 8, and 24 h. The OD600 nm of the samples from three independent cultures was measured, and the averaged values from each time point were plotted. Wild-type and TF0022-ko cells were harvested after 5 days of culture, which corresponded to the late logarithmic or early stationary growth phase. After being treated with

10% trichloroacetic acid, DOK2 cells were lysed with a cell lysis solution (420 mg mL−1 urea, 152 mg mL−1 thiourea, 80 mg mL−1 3-[(3-chloramidopropyl) dimethylammonium]-1-propanesulfonate, 1 mM EDTA, 0.2% tributylphosphine, 40 mM Tris-HCl, pH 8.0). Each total protein sample was separated by a rehydrated Immobiline DryStrip (pH 4–7, 13 cm, GE Healthcare, Little Chalfont, Buckinghamshire, UK), followed by fractionation by sodium dodecyl sulfate-12% PAGE. The Coomassie Blue R-250 (CB)-stained gels were scanned with an Image Scanner (Amersham Biosciences, Uppsala, Sweden). Protein bands excised from the CB-stained gels were destained with 25 mM NH4HCO3 buffer containing 30% CH3CN, dehydrated with 100% CH3CN, reduced with 10 mM dithiothreitol in 25 mM NH4HCO3 for 1 h at 56 °C, and subsequently alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 for 45 min in the dark. Samples were dehydrated and digested with 10 ng μL−1 sequencing grade trypsin (Promega Co., Madison, WI) in 25 mM NH4HCO3 overnight at 37 °C. Peptides were extracted with 5% trifluoroacetic acid in 50% CH3CN for 1 h, spotted onto a matrix-assisted laser desorption/ionization (MALDI) target plate in combination with CHCA matrix (Sigma-Aldrich Co., St.