Human stool collection
Stool samples from an adult healthy human donor were collected using a commercially available stool collection vessel and with Northeastern University IRB approval. Within 5 min of collection, 1 g of stool was resuspended in 9 mL of sterile 20% glycerol in phosphate-buffered saline (PBS) and homogenized for 30 s using a vortex. One milliliter aliquots of this mixture was loaded in cryotubes and stored at − 80 °C. For cultivation experiments, individual aliquots of the frozen stool samples were then removed from the freezer and immediately transferred to a Coy Anaerobic Vinyl chamber with an atmosphere of 5% hydrogen, 10% CO2, and 85% nitrogen.
Isolation and cultivation of helper-dependent bacteria
Serial dilutions of feces samples were spread-plated onto various rich medium (listed below). Plates were then incubated at 37 °C. All experiments were performed under strictly anaerobic conditions. Two different methods were used to obtain helper-dependent organisms: method A. The time of formation for all colonies were tracked for a week. Late-forming colonies (3–7 days) were diluted and spread-plated on fresh medium and nearby (< 5 mm), early-forming colonies (appearance after 1–2 days) were then resuspended in PBS at a high density and spotted (5 μL) on the sample plate. The co-cultures were incubated for up to 1 week in the chamber and observed daily. Growth induction of the dependent organism around the spotted helper indicated a positive hit. Method B: After spread-plating the feces samples, a helper spot of Escherichia coli K12 BW25113 was spotted on the same plate (5 μL). After incubation, colonies were picked in close proximity to the E. coli spot and tested for dependency on E. coli in the same way described for method A.
Growth media
The following growth media were used in this study: brain heart infusion (BHI) supplemented with 5 g/L yeast extract, 1 g/L cysteine, and 15 mg/L hemin (only in agar) (BHIych); LYHBHI: BHI supplemented with 5 g/L yeast extract, 0.5 g/L cysteine, 5 mg/L hemin, 1 g/L cellobiose, 1 g/L maltose [16]; fastidious anaerobe agar (FAA); FAA with 5% (v/v) defibrinated sheep blood added (FAA blood); YCFAG (per liter): 4.5 g glucose (anhydrous), 10 g casitone, 2.5 g yeast extract, 4 g NaCHO3, 1 g cysteine, 0.45 g K2HPO4, 0.45 g KH2PO4, 0.9 g NaCl, 0.09 g MgSO4x7H2O, 0.09 g CaCl2, 1 mg resazurin, 10 mg hemin, 10 μg biotin, 10 μg hydroxycobalamin, 30 μg para-aminobenzoic acid, 50 μg folic acid, 150 μg pyridoxamine, 33 mM acetate, 9 mM propionate, 1 mM isobutyrate, 1 mM isovalerate, 1 mM valerate, 50 μg thiamine, 50 μg riboflavin, 15 g agar (optional)) [31]. Heat-labile vitamins were added after the medium was autoclaved. All media were prereduced overnight in an anaerobic chamber.
Taxonomic assignment by 16S rRNA gene sequencing
PCR was performed using the general bacterial primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3′) to amplify part of the 16S rRNA gene. The PCR reaction mixture was 12.5 μL GoTaq Master Mix (Promega), 1 μL 10 μM 27F and 1492R primers (Operon), 9.5 μL Nuclease Free Water (Promega), and 1 μL of a colony resuspended in 100 μL sterilized distilled water. PCR was performed using an Eppendorf Mastercycler Personal, and the amplification conditions were one cycle of 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s; and finally one cycle of 72 °C for 7 min. Amplification of PCR reactions were confirmed using gel electrophoresis on a 0.8% agarose gel containing ethidium bromide. Successful PCRs were sequenced by Macrogen Corporation using the 27F primer using the Applied Biosystems 3730xl DNA analyzer. Quality control for sequences was performed using DNA Baser (www.DnaBaser.com), in which ends were trimmed until there were more than 75% good bases (defined by having a QV score of higher than 25) in an 18-base window. Identification of phylogenetic neighbors and calculations of pairwise sequence similarity were done using the EZTaxon server (http://www.eztaxon.org). 98.7% similarity by 16S was used to determine species level cutoff using a previously suggested level [32].
Screening E. coli deletion mutants for a lack of growth induction
For the library screen, a selection of 283 strains from the E. coli small-, medium-, and large-scale deletion libraries were screened [20, 21]. The mutants were obtained from the Japanese National BioResource Project. The E. coli strains were grown anaerobically overnight in BHIych. The next day, KLE1255 was plated onto BHIych agar and 1 μL spots of E. coli cultures were added to the plates. After 2 days of incubation, plates were evaluated for growth of KLE1255. All other induction assays were performed using E. coli suspensions, prepared in phosphate-buffered saline or growth medium, rather than overnight cultures as no difference was observed for the different treatments.
Quinone induction experiments
Inoculums of strains were either created by picking colonies from agar plates seeded with E. coli K12 BW25113 or from 24 to 48 h cultures in BHIych. These inocula were then bead spread on solid BHIych until dried, and 5 μL of a 1-mM stock of DHNA, MK-4, or menadione (all purchased from Sigma and resuspended in 100% ethanol) was spotted, with ethanol as a control. For Faecalibacterium sp. KLE1255, concentrations of up to 10 mM of DHNA, MK-4, or menadione were also tested. An expanded panel of quinones were also tested for Faecalibacterium sp. KLE1255 induction: MK-8 purified from E. coli K12 BW25113, and MK-4, MK-5, MK-6, MK-7, and MK-8 purified from Micrococcus luteus KLE1011. Ubiquinones tested include Q1, Q2, Q4, Q9, and Q10 (all available from Sigma-Aldrich) as well as Q7 and Q8, which were purified from E. coli. These were tested using multiple solvents (acetone, MeOH, DMSO, and hexane) at concentrations of 10 mg/mL, 1 mg/mL, and 0.1 mg/mL and 10 μL spotted. Methods for isolation of the expanded quinone panel are described below.
Isolation of quinones
For the expanded quinone panel, quinones were isolated from Escherichia coli K12 BW25113 and Micrococcus luteus KLE1011. Strains were streaked on plates of BHIych or R2Asea, respectively, and individual colonies were picked to inoculate 5.0 mL cultures, which were grown for 24 h at 250 rpm. These starter preps were used to inoculate 1.0 L cultures, which were grown for 24 h at 160 rpm. A batch of eight, 1.0 L cultures was routinely prepared for each organism for cell pellet and supernatant extractions. One liter cultures of bacteria were centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge equipped with a JLA 8.1000 rotor at 4 °C. Cells were pelleted at 4500 rpm (5053×g) for 20 min. The supernatant was poured off and the cells were washed with 1% NaCl. The cells were pooled and pelleted at 6000 rpm (8983×g) for 20 min. The supernatant was discarded, and the cells were submitted to quinone extraction. Quinones were isolated either via extraction of saponified cells or via direct solvent extraction of pelleted cells. Saponification was performed in line with established protocols [33]. Briefly, pelleted cells (from 8 × 1.0 L preps) were suspended in a 150 mL, 3:2:1 mixture of ethanol:water:25% KOH containing 2.5 g pyrogallol, transferred to a round bottom flask and refluxed for 20 min at 100 °C under an inert atmosphere of dry gas (nitrogen or argon) and under the exclusion of light while stirring vigorously. The mixture was immediately cooled to room temperature in an ice-water bath and then extracted 4× with equivolume portions of heptane. The organic layers were pooled, dried over anhydrous Na2SO4, filtered, and concentrated to dryness in vacuo. The dried, oily material was stored under inert gas (nitrogen or argon) at − 20 °C until chromatographic purification was performed. Direct solvent extraction was performed by suspending pelleted cells (from 8 × 1.0 L preps) in 300 mL of a 3:1 ethanol:diethyl ether solution, transferring to a round bottom flask and stirring vigorously for 2.5 h under an atmosphere of dry, inert gas and under the exclusion of light. The suspension was filtered, the cells were rinsed with a portion of diethyl ether, and the filtrate was dried to 25% its original volume in vacuo. The concentrate was diluted with water and extracted 3× with 500-mL portions of heptane. The pooled organic layer was then dried and stored as described above. Quinones and menachromenols were purified using an Agilent Technologies 1200 Series High Performance Liquid Chromatography system equipped with G1361A Prep Pumps and a G1315D diode array detector. Samples were prepared in 2.0 mL of acetonitrile and were purified on a Phenomenex Luna C8(2), 250 × 21.20 mm, 5 mm reverse phase HPLC column. Material was eluted using an isocratic solvent system consisting solely of HPLC grade acetonitrile at a 10-mL min−1 volume flow rate.
NMR characterization of isolated quinones
All NMR spectra of bacterially derived compounds were acquired using a CDCl3 susceptibility matched 5-mm Shigemi® NMR tube on a Varian VNMRS 600-MHz NMR spectrometer equipped with a 5-mm HCN AutoX inverse probe. Data was acquired using VnmrJ version X software and was analyzed using MestReNova version 7.0.0-8331 software. Trace acid was removed from “100%” CDCl3 solvent (99.96 atom % D–Aldrich) by passing over a plug of Brockmann grade I activated, basic aluminum oxide (Aldrich) immediately before use. Spectra were referenced to TMS or residual protio solvent.
Isolation of genomic DNA from Faecalibacterium sp. KLE1255 for whole genome sequencing
A phenol/chloroform prep was performed to obtain genomic DNA. Three milliliter of a turbid culture were pelleted and resuspended in 500-μL lysis buffer (20 mM Tris 7.5; 50 mM EDTA; 100 mM NaCl). Fifty microliter of 20 mg/mL freshly prepared lysozyme and 100 μg/mL proteinase K were added and the mixture. After 30–60-min incubation, 60 μL 10% (w/v) sarkosyl (N-lauroylsarkosine) was added and vortexed. Next, 600 μL TE-saturated phenol was added; the contents vigorously vortexed for 15 s and centrifuged for 5 min at 13,000 rpm. Then, the aqueous phase was removed to a new tube and 600 μL phenol/chloroform were added, the mixture vortexed and the aqueous phase removed to a new tube again. One-tenth volume (60 μL) of 3 M NaOAc was added, mixed, and two volumes EtOH were added. The tube was inverted until the DNA precipitated (4–6 times). The DNA was centrifuged down for 1 min and the supernatant removed. One-hudred fifty microliter of 70% (v/v) EtOH were added and vortexed, and the DNA centrifuged again. The supernatant was removed and the pellet air-dryed for 10–15 min. Finally, the DNA was resuspended in 100 μL elution buffer (10 mM Tris-Cl, ph 8.5).
Whole genome sequencing and annotation of Faecalibacterium sp. KLE1255
Five microliter of genomic DNA was sent to George Weinstock at The Genome Institute at Washington University, St. Louis, Missouri, for Illumina sequencing. The draft genome, consisting of 119 contigs, was annotated using the RAST (Rapid Annotation using Subsystem Technology) server [34] and the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.