Achromopeptidase

Pulsed-field gel electrophoresis (PFGE) plug was prepared using the CHEF Bacterial Genomic DNA Plug Kit (BioRad, CA, USA), replacing lysozyme with achromopeptidase (Wako, Osaka, Japan) for bacterial lysis. The plug was treated with a restriction enzyme or S1 nuclease, followed by PFGE (1% agarose gel; 0.5× TBE; 6 V/cm; pulse-time, 2.2–65.0; angle, 120°; run time, 20 h) [16 (link)].

Isolates were cultured overnight in screw-capped glass tubes (Pyrex, Iwaki Glass, Tokyo, Japan) filled with Todd-Hewitt broth (BD Biosciences, San Jose, CA, USA) and supplemented with 0.2% yeast extract (BD Biosciences) (THY) at 37°C in an ambient atmosphere. Bacterial cells were lysed with 10 units/mL mutanolysin (Sigma, St. Louis, MO, USA), 10 mg/mL lysozyme (Wako, Osaka, Japan), and 0.5 mg/mL achromopeptidase (Wako), and genomic DNA was extracted from overnight cell cultures using a Maxwell^® RSC instrument with a Maxwell^® RSC PureFood GMO and Authentication Kit (Promega Corporation, Madison, WI, USA). Paired-end libraries were generated from extracted DNA with a Nextera^® XT DNA kit (Illumina, San Diego, CA, USA). Libraries were sequenced with Illumina instruments (HiSeq X and Miseq) and paired-end sequence reads (150 bp and 301 bp, respectively) were obtained.

Fecal samples were freshly collected and transported to the laboratory under anaerobic condition in AnaeroPack (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) at 4°C. The fecal samples were frozen by liquid nitrogen in phosphate-buffered saline containing 20% glycerol, and stored at -80°C until use. Bacterial DNA was extracted from the fecal samples by enzymatic lysis method using lysozyme (Sigma-Aldrich Co., St. Louis, Missouri) and achromopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan), as previously described [16 , 17 (link)].

To extract fecal bacterial DNA, we used an enzymatic lysis method with lysozyme (Sigma-Aldrich Co., St. Louis, MO) and achromopeptidase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) for all subjects, as described previously^{13 (link)}. To amplify the bacterial 16S rRNA gene V3–V4 regions, we used the 16S amplicon PCR forward primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and the 16S amplicon PCR reverse primer (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′), with adaptor sequences for Illumina indexing. PCRs were run for 25 cycles, using the KAPA HiFi HotStart ReadyMix PCR Kit (Nippon Genetics Co., Ltd, Tokyo, Japan). Amplicons were purified with AMPure^® XP magnetic purification beads (Beckman Coulter, Inc., Brea, CA) and quantified with 4200 TapeStation (Agilent Technologies Japan, Ltd., Tokyo, Japan). Equal amounts of amplicons from all the samples were sequenced with the MiSeq System (Illumina, Inc., Tokyo Japan), according to the manufacturer’s instructions^{13 (link)}. In the control group, we re-analyzed the 16S rRNA sequence at the V3-V4 region in the same way as for the bowel prep group from preserved DNA samples and did not use any previously obtained data^{13 (link)}.

DNA extraction from feces was performed according to the literature with minor modifications [40 (link),41 (link)]. Bacterial pellets from feces were suspended with a 10 mM Tris-HCl/10 mM EDTA solution. The sample suspension was incubated with 15 mg/mL lysozyme (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 37 °C for 1 h. A final concentration of 2000 units/mL of purified achromopeptidase (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) was added and then incubated at 37 °C for 30 min. The suspension was added at 1% (w/v) sodium dodecyl sulfate and 1 mg/mL proteinase K (Merck KGaA) and incubated at 55 °C for 1 h. After centrifugation, the bacterial DNA was purified using a phenol/chloroform/isoamyl alcohol (25:24:1) solution. The DNA was precipitated by adding ethanol and sodium acetate, and RNase treatment and polyethylene glycol (PEG) precipitation were performed.

Mouse feces (4 mice/group) were collected at weeks 2 and 5 by means of fecal disimpaction. The collected fecal samples were immediately mixed with an appropriate volume of RNAprotect Bacteria Reagent (Qiagen, Tokyo Japan) and stored at –80°C until analysis. Bacterial genomic DNAs were extracted as follows. After thawing, the fecal samples were subjected to centrifugation at 8000 x g for 10 min. The precipitate was suspended in 200 μl of 10 mM Tris-HCl/1 mM EDTA (TE) buffer, pH 7.0, containing 15 mg/mL lysozyme (Sigma-Aldrich, St. Louis, MO, USA). After incubation of the mixture at 37°C for 1 h, 200 μl of achromopeptidase (4000 units/mL, FUJIFILM Wako Pure Chemical) were added, and the mixture was further incubated at 37°C for 30 min. Subsequently, 400 μl of a mixture containing 2 mg/mL of proteinase K and 2% sodium dodecyl sulfate were added, and the resulting mixture was incubated at 55°C for 1 h. The mixture was then treated with phenol/chloroform/isoamyl alcohol (Life Technologies Japan, Tokyo, Japan), followed by treatment with RNase A (FUJIFILM Wako Pure Chemical), as described previously [12 (link)]. DNA was precipitated by adding an equal volume of a mixture of 20% polyethylene glycol 6000 and 2.5 M NaCl followed by centrifugation at 8000 x g at 4°C, washed with 75% ethanol, and dissolved in TE.