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Supplementary Materials for The Genetic Basis for the Cooperative Bioactivation of Plant Lignans by a Human Gut Bacterial Consortium Elizabeth N. Bess1, Jordan E. Bisanz1, Peter Spanogiannopoulos1, Qi Yan Ang1, Annamarie Bustion1, Seiya Kitamura2, Diana L. Alba3, Dennis W. Wolan2, Suneil K. Koliwad3, Peter J. Turnbaugh1,4* *Correspondence to: [email protected] This PDF file includes: Materials and Methods Figures S1–S3 Tables S1–S11 1 Materials and Methods Bacterial culturing conditions All bacteria were cultured at 37 °C in an anaerobic chamber (Coy Laboratory Products) with an atmosphere composed of 2-3% H2, 20% CO2, and the balance N2. Culture media was composed of Bacto Brain Heart Infusion (BD Biosciences, 37 g/L) supplemented with L-cysteine-HCl (0.05%, w/v), L-arginine (1%, w/v), menadione (1 µg/mL), and hemin (5 µg/mL) (referred to herein as BHI++) and allowed to equilibrate in the anaerobic environment prior to use. Bacterial strains The bacterial strains in our Coriobacteriia collection have previously been extensively described (1). Each of these strains has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection; draft genomes are available in NCBI’s BioSample database. Organism BioSample accession Eggerthella lenta 32-6-I 6 NA SAMN08365966 Eggerthella lenta DSM 2243 SAMN08365978 Eggerthella lenta 11C SAMN08365960 Eggerthella lenta RC4/6F SAMN08365982 Eggerthella lenta FAA1-1-60AUCSF SAMN08365980 Eggerthella lenta AN51LG SAMN08365970 Eggerthella lenta 14A SAMN08365961 Eggerthella lenta AB12 #2 SAMN08365968 Eggerthella lenta 28B SAMN08365965 Eggerthella lenta Valencia SAMN08365983 Eggerthella lenta DSM 15644 SAMN08365977 Eggerthella lenta DSM 11863 SAMN08365976 Eggerthella lenta CC7/5 D5 2 SAMN08365972 Eggerthella lenta CC8/2 BHI2 SAMN08365973 Eggerthella sinensis DSM 16107 SAMN08365985 Eggerthella lenta W1 BHI 6 SAMN08365984 Eggerthella lenta 22C SAMN08365964 Eggerthella lenta AB8 #2 SAMN08365969 Eggerthella lenta CC8/6 D5 4 SAMN08365974 Eggerthella lenta DSM 11767 SAMN08365975 Eggerthella lenta 1-3-56 SAMN02463797 Eggerthella lenta MR1 #12 SAMN08365981 Gordonibacter pamelaeae 3C SAMN08365986 Gordonibacter sp. 28C SAMN08365987 Paraeggerthella hongkongensis RC2/2 A SAMN08365988 2 Blautia producta DSM 3507 and Lactonifactor longoviformis DSM 17459 were purchased from DSMZ; draft genomes are available in NCBI’s BioSample database. Organism BioSample accession Blautia producta DSM 3507 SAMN08397823 Lactonifactor longoviformis DSM 17459 SAMN08397824 Chemicals Pinoresinol, lariciresinol, and secoisolariciresinol were obtained from Separation Research Ltd. Enterodiol, enterolactone, and corticosterone were obtained from Sigma Aldrich. Culturing the Coriobacteriia strain collection with PINO Wells of 96-well plates containing 99 µL of BHI++ media supplemented with PINO (510 µM) were inoculated (1:100), in triplicate, from dense cultures of each of the 25 strains of our Coriobacteriia collection. Wells were also prepared to serve as sterile controls. Plates were sealed with tape to minimize evaporation and incubated at 37 °C for 48 hours. Next, the plates were centrifuged at 2000 rpm for 10 min at 4 °C, and the supernatant was harvested and stored at -20 °C. Samples were thawed prior to performing analyte measurements via HPLC, using HPLC Method A (described below). Results are summarized in Table S2. Culturing bacteria with lignans for time-course, RNA-Seq, and qRT-PCR experiments Culturing E. lenta DSM 2243 with PINO Hungate tubes containing 5 mL of BHI++ were inoculated (1:100), in pairs, from dense cultures of E. lenta DSM 2243. Sterile controls were also prepared. Culture tubes were loosely capped and incubated at 37 °C until mid-log-phase growth was achieved, at which time pairs of cultures were exposed to either 50 µL of pinoresinol (PINO, 50 mM in methanol) or 50 µL of vehicle (methanol). Then, 1.5 hours following PINO (n=3 cultures) or vehicle (n=3 cultures) exposure, cultures were removed from the anaerobic chamber and centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was decanted, and the remaining cell pellet was either flash-frozen in liquid nitrogen and subsequently stored in a -80 °C freezer until RNA was extracted for qRT- PCR analysis or 1 mL of TRI Reagent (Sigma Aldrich) was added to the pellet for immediate extraction of RNA for RNAseq (see below for details of this procedure). Additional cultures differentially exposed to PINO (n=3) or vehicle (n=3) were maintained in the anaerobic chamber. Throughout the course of incubating these cultures, OD600 was periodically measured using a Hach spectrophotometer (model DR1900) that was housed in an anaerobic chamber. Following addition of PINO or vehicle, each culture was periodically sampled (100 µL aliquots); supernatant was collected and stored at -20 °C. Samples were thawed prior to HPLC analysis, which was performed according to HPLC Method A (described below). Culturing B. producta DSM 3507 with SECO Hungate tubes containing 5 mL of BHI++ were inoculated (1:100), in pairs, from dense cultures of B. producta DSM 3507. Sterile controls were also prepared. Culture tubes were loosely 3 capped and incubated at 37 °C until mid-log-phase growth was achieved, at which time pairs of cultures were exposed to either 50 µL of secoisolariciresinol (SECO, 50 mM in methanol) or 50 µL of vehicle (methanol). Then, ~1.5 hours following SECO (n=3 cultures) or vehicle (n=3 cultures) exposure, cultures were removed from the anaerobic chamber and centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was decanted, and the remaining cell pellet was flash- frozen in liquid nitrogen and subsequently stored in a -80 °C freezer until RNA was extracted for analysis via qRT-PCR or RNAseq (see below for details of this procedure). To assess consumption of SECO over time, cultures (100 µL of BHI++) were prepared in a 96- well plate. Five sets of three cultures and one sterile control, each exposed to SECO (500 µM), were prepared. Following 2, 5, 7, 10, and 25 hrs of incubation at 37 °C, one set was of cultures was harvested; supernatant was collected and stored at -20 °C. Samples were thawed prior to HPLC analysis, which was performed according to HPLC Method B (described below). Culturing G. pamelaeae 3C with dmSECO Hungate tubes containing 5 mL of BHI++ and 500 µM of SECO (50 µL from a stock solution of 50 mM SECO in methanol) were inoculated (1:100), in pairs, from dense cultures of B. producta DSM 3507. Sterile controls were also prepared. Culture tubes were loosely capped and incubated at 37 °C. After 22.5 hours of incubation, HPLC analysis of supernatant (using HPLC Method B) demonstrated that SECO had been fully consumed. Maintaining cultures in an anaerobic chamber, the cultures were partitioned into microcentrifuge tubes and centrifuged to pellet the cells. The supernatant was passed through a 0.2 µm filter to sterilize the spent media, which contained the metabolic byproduct of SECO, presumably didemethylsecoisolariciresinol (dmSECO). Concurrent to preparation of these cultures of B. producta DSM 3507, hungate tubes containing 3 mL of BHI++ were inoculated (1:100), in pairs, from dense cultures of G. pamelaeae 3C. Sterile controls were also prepared. Culture tubes were loosely capped and incubated at 37 °C until mid-log-phase growth was achieved, at which time pairs of cultures were exposed to 2 mL of the sterilized, spent media that was harvested from the incubation of B. producta DSM 3507 with either SECO (n=3 cultures) or vehicle (n=3 cultures). The spent, filter-sterilized media from B. producta DSM 3507 was also added to sterile controls to monitor sterility of the spent media. After 1.5 hours, the cultures of G. pamelaeae 3C were removed from an anaerobic chamber and centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was decanted, and the remaining cell pellet was flash-frozen in liquid nitrogen and subsequently stored in a -80 °C freezer until RNA was extracted for RNAseq (see below for details of this procedure). Prior to removing the cultures from the anaerobic chamber, a 400 µL aliquot was obtained of each of the cultures of G. pamelaeae 3C that had been exposed to spent, filter-sterilized media that was generated from the incubation of B. producta DSM 3507 with SECO. These aliquots, maintained in an anaerobic chamber, were incubated for an additional 21.5 hours. Supernatant was evaluated by HPLC (HPLC Method B) to assess production of END by G. pamelaeae 3C. No END was detected in the sterile control. 4 During the course of the work reported herein, we obtained a pure sample of dmSECO (synthetic methods reported below). This material was used to validate by qRT-PCR the results of the RNAseq analysis from the incubation of G. pamelaeae 3C with spent, filter-sterilized media that was generated from the incubation of B. producta DSM 3507 with SECO or vehicle. Hungate tubes containing 5 mL of BHI++ were inoculated (1:100), in pairs, from dense cultures of G. pamelaeae 3C. Sterile controls were also prepared. Culture tubes were loosely capped and incubated at 37 °C until mid-log-phase growth was achieved, at which time pairs of cultures were exposed to either 50 µL of dmSECO (50 mM in methanol) or 50 µL of vehicle (methanol). Then, 1.5 hours following dmSECO (n=3 cultures) or vehicle (n=3 cultures) exposure, cultures were removed from an anaerobic chamber and centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant was decanted, and 1 mL of TRI Reagent was added to the remaining cell pellet for immediate extraction of RNA (see below for details of this procedure), which was subjected to a qRT-PCR assay. Additional cultures differentially exposed to dmSECO (n=3) or vehicle (n=3) were maintained in the anaerobic chamber. During incubation, OD600 was periodically measured using a Hach spectrophotometer (model DR1900) that was housed in the anaerobic chamber.