Characterization of strain 14 T

2.2.3.1 Gram staining

Gram staining was carried out using the method described by Bartholomew and Mittwer (1952).

Crystal violet solution

Solution A was prepared by adding 2 g of crystal violet (dye content ≥ 90 %) into 20 ml of 95 % (v/v) ethanol. Solution B was prepared by dissolving 0.8 g of ammonium oxalate in 80 ml of distilled water. Solution A and B were mixed and stored in dark for 24 h before use.

Gram iodine solution

1 g of iodine and 2 g of potassium iodide were finely grinded using a mortal and a pestle. 300 ml of distilled water was slowly added until iodine completely dissolved. Solution was kept in an amber bottle until use.

Decolourizing agent

50 ml of 95 % (v/v) ethanol and 50 ml of 100 % (v/v) acetone were mixed.

Safranin solution

A stock solution of safranin was prepared by dissolving 2.5 g of safranin O in 100 ml of 95 % (v/v) ethanol. A working solution was prepared by diluting 10 ml of the stock solution in 90 ml of distilled water.

Gram staining method

A small volume of bacterial suspension was spread on a glass microscope slide and air-dried. Air- dried culture was fixed by passing the slide briefly over flame. The slide was flooded with crystal violet solution for 1 min and washed with a gentle stream of tap water. The slide was then covered with Gram iodine solution for 1 min and washed with tap water. Cells were briefly washed with Decolourizing agent and drained well. Safranin solution was used to counterstain for 30 sec. The slide

was washed with tap water and drained using a paper towel. The cells were observed at 1,000× power magnification using immersion oil mount using Olympus CX21 microscope.

2.2.3.2 Endospore staining Malachite green stain solution

0.5 g of Malachite green was dissolved in 100 ml of distilled water. The solution was kept in the dark overnight before use.

Endospore staining method

Cells of strain 14T _{were grown in 9 ml mineral medium containing 0.5 % (w/v) citrus pectin. Cells} were incubated at 37℃ for 31 days with a constant shaking. Endospore formation was examined by Schaeffer-Fulton staining method (Schaeffer and Fulton, 1933). A small volume of bacterial

suspension was spread on a glass microscope slide. The suspension was air-dried and heat fixed over Bunsen flame. A piece of blotting paper was used to cover the slide which was then saturated with Malachite green stain solution. The slide was steamed for 5 min over boiling water while gentling dripping Malachite green solution to keep the paper moist. The slide was washed with tap water, followed by counterstaining with Safranin solution (from Gram staining method) for 30 sec. The slide was washed with tap water and drained using a paper towel. Cells were observed under the oil

immersion lens (1,000×) using Olympus CX21 microscope for the presence of endospores.

2.2.3.3 Cell motility by phase contrast microscopy

A small volume of 1-week-old bacterial culture was added onto a glass microscope slide. A glass coverslip was placed on top of the cell suspension. Excess liquid was dried using a paper towel. Cell motility was observed under phase contrast with a DM2500 microscope at x400 magnification (Leica Microsystems, Germany). Digital images of cells were captured using Leica Application suite software.

2.2.3.4 Transmission electron microscopy (TEM) Imaging free cells

Cells of strain 14T were grown at 37℃ for 96 h in 9 ml mineral medium containing 0. 5 % (w/v) D- fructose. Cells were pelleted by centrifugation at 10,000 g for 5 min at room temperature. Cells were fixed, stained, prepared as ultra-thin sections for TEM, and imaged at Manawatu Microscopy & Imaging Centre (Palmerston North, New Zealand). Pelleted cells were fixed in modified Karnovsky’s fixative (3 % glutaraldehyde (v/v) 2 % formaldehyde (w/v) in 0.1 M phosphate buffer (pH 7.2)) for 2 h at room temperature. Fixed cells were pelleted by centrifuging at 4,000 g for 4 min, and the fixatives were drained and discarded. 3 drops of 20 % (w/v) bovine serum albumin (BSA) were added to the pellet. Cells were centrifuged again at 4,000 g for 4 min. BSA was coagulated by adding 1 drop of 25 % (w/v) glutaraldehyde and leaving at room temperature for 5 min. Pellets were cut into thin slices and rinsed three times in 0.1 M phosphate buffer (pH 7.2) for 10 min. Cells were post fixed in 1 % (w/v) osmium tetroxide dissolved in 0.1 M phosphate buffer (pH 7.2) for 30 min at room temperature. Cells were rinsed three times in 0.1 M phosphate buffer (pH 7.2) before being dehydrated in

increasing concentrations of acetone (25 % (v/v), 50 % (v/v), 75 % (v/v), 95 % (v/v), and 100 % (v/v)) for 10 min each. Cells were further dehydrated by incubating in 100 % (v/v) acetone twice for 1 h each. Cells were stirred overnight in 50:50 (v/v) resin:acetone mix (ProSciTech, Australia). Once the acetone evaporated off, fresh 100 % (v/v) resin (Procure812, ProSciTech, Australia) was added and the cells were stirred for 8 h. This step was repeated twice. Samples were embedded with fresh 100 % (v/v) resin, and cured in a 60 ℃ oven for 48 h. The embedded block of cells was sliced into ultra-thin sections (100 nm) using a diamond knife (Diatome, Austria) on Ultramicrotome (Leica Microsystems, Germany). Sections were stretched over a copper grid and stained with saturated uranyl acetate in 50 % (v/v) ethanol for 4 min. Grids were washed with 50 % (v/v) ethanol and MilliQ water, followed by a further staining in lead citrate for 4 min. Samples were viewed using FEI Tecnai G2 Spirit BioTWIN (FEI Company, Czech Republic).

Bacterial adhesion to the cell walls of orange peel and kiwifruit

Orange and kiwifruit were purchased from a local supermarket. The surface of fruit was wiped using 80 % (v/v) ethanol before placing them in a UV-sterilized Infinity Class II biosafety cabinet (ESCO). Using a sterile razor blade, orange peel and kiwifruit were cut into 0.5-1 mm slices. Fruit sections were transferred to a UV-sterilized anaerobic chamber inside sterile plastic containers. Using sterile forceps, 5 – 10 pieces of fruit sections were dispensed into N2-washed sterile Hungate tubes that were then sealed with screw caps and rubber stoppers. The tubes were topped with 9 ml of mineral medium which was then inoculated with 1-week-old bacterial cultures. Fruit sections were collected after 72 h of incubation at 37℃ with a constant shaking. Sections were washed briefly with anaerobic PBS and immediately submerged in modified Karnovsky’s fixative. TEM imaging was carried out as above without the centrifugation procedures.

2.2.3.5 Genomic DNA extraction TES buffer (pH 8.0)

0.254 g of Tris-HCl; 0.048 g of Tris; 0.116 g of NaCl; and 0.068 g of EDTA were dissolved in 400 ml of distilled water. The solution was sterilized by autoclaving.

Lysozyme solution

200 mg of lysozyme from chicken egg white (≥ 40,000 U/mg; Sigma) were dissolved in 10 ml of autoclaved water. The solution was dispensed into 1 ml microcentrifuge tubes in 250 µl aliquots, and stored at -20℃ until use.

Proteinase K solution

200 mg of proteinase K from Tritirachium album (41 U/mg; Sigma) were dissolved in 10 ml of autoclaved water. The solution was dispensed into 1 ml microcentrifuge tubes in 250 µl aliquots, and stored at -20℃ until use.

RNase solution

200 mg of ribonuclease A from bovine pancreas (≥ 50 Kunitz U/mg; Sigma) were dissolved in 10 ml of autoclaved water. 250 µl aliquots of the solution were kept at -20℃ until use.

TNS solution (pH 8.0)

500 mM Tris-HCl; 100 mM NaCl; and 10 % (w/v) SDS were dissolved in distilled water by autoclaving.

PEG solution

1.6 M NaCl was dissolved in distilled water. 30 % (w/v) polyethylene glycol 6000 was added and dissolved by autoclaving.

EB buffer

10 mM Tris-HCl was dissolved in distilled water. 2M NaOH was used to adjust the pH to 8.5. The solution was sterilized by autoclaving.

Sodium acetate solution

3 M sodium acetate was dissolved in distilled water. Using 2M HCl, the pH of the solution was adjusted to 5.2. Solution was sterilized by autoclaving.

Phenol-chloroform extraction of genomic DNA

Bacterial cell pellets were obtained from 1-week-old cultures grown on RC medium containing 1 % (w/v) D-fructose. Cells were suspended in 0.5 ml of TES buffer and mixed vigorously. 10 µl of lysozyme was added to the cell suspension followed by vigorous mixing. Suspension was incubated in a heat block at 37℃ for 2 h. Samples were immediately transferred on ice. 10 µl of proteinase K and 10 µl of RNase were added to the suspension. The mixture was vortexed and incubated in a water bath at 65℃ for 1 h. 100 µl of TNS solution was added and samples were further incubated at 65℃ for 30 min. Samples were transferred to 2 ml clean Eppendorf tubes. 700 µl of

phenol/chloroform/isoamylalcohol (25:24:1, pH8; Sigma) was added and mixed with the sample by gentle inversion. Samples were centrifuged at 20,000 g for 20 min at 4℃. 500 µl of upper layer was carefully pipetted and transferred into a fresh 2 ml Eppendorf tube. 500 µl of

chloroform/isoamylalcohol (24:1; Sigma) was added and mixed with the sample by gentle inversion, followed by centrifugation at 20,000 g for 5 min at 4℃. 400 µl of upper layer was carefully pipetted and transferred into a clean 2 ml tube. 800 µl of PEG solution was added to the sample and mixed by

gentling inverting the vial 5 times. Precipitated DNA was centrifuged at 20,000 g for 1 h at 4℃. Liquids were carefully poured out and 500 µl of ice cold 70 % (v/v) ethanol was added to wash the pellet. Precipitates were centrifuged at 20,000 g for 10 min at 4℃. The supernatant was carefully poured out and ethanol-wash step was repeated. Ethanol was carefully poured out and the tubes were kept inverted on a paper towel to air-dry the pellet. DNA precipitates were eluted in 50 µl EB buffer, and left to dissolve in the fridge (4℃) overnight. DNA concentration and purity were checked using Nanodrop®_{-1000. DNA samples were further purified and concentrated using ethanol precipitation} method.

Ethanol precipitation of DNA

Extracted DNA samples were combined and the volumes of DNA samples were carefully measured and recorded. 1/10 volume of sodium acetate solution was added to the DNA samples and mixed by gentle inversion. 2.5 volume of ice cold 100 % (v/v) ethanol was added and mixed by gentle

inversion. The mixture was stored at -80℃ overnight. The mixture was centrifuged at 20,000 g for 15 min at 4℃, and the supernatant was carefully poured out. The pellet was washed with 1 ml of ice cold 70 % (v/v) ethanol. Precipitated DNA was centrifuged at 20,000 g for 10 min at 4℃, and ethanol was carefully decanted. Ethanol-wash step was repeated and supernatant was carefully poured out. Tubes were kept upside down on a paper towel to air-dry the pellets. Pellets were eluted in 20 µl of EB buffer and left to dissolve overnight at 4℃.

Quantification of DNA

DNA quantification was carried out using Qubit™ dsDNA BR Assay Kit (Invitrogen) according to the manufacturer’s instructions.

2.2.3.6 Agarose gel electrophoresis TAE buffer (1×)

50× TAE stock solution was prepared by mixing 2 M Tris; 50 mM EDTA; and 60 ml of glacial acetic acid in 940 ml of distilled water. 2M NaOH was used to adjust pH to 8.0 of necessary. Stock solution was diluted 50× in distilled water to make 1× TAE buffer.

SYBR® _{Green solution}

SYBR® _{Green I Nucleic Acid Gel Stain was purchased from Invitrogen. 100 µl of dye was mixed with} 900 µl of 1× TAE buffer (2.6.7.1). Aliquots were stored at -20℃ until use. 100 µl of dye-buffer mixture was added per 100 ml of agarose gel.

Orange G loading dye (10×)

100 mg of orange G and 15 ml glycerol were dissolved in 35 ml of distilled water. The solution was sterilized by autoclaving and stored at -20℃ until use. 1 µl of loading dye was used per 5 µl of DNA sample.

Gel electrophoresis

1 % (w/v) agarose gel was prepared with 1× TAE buffer. DNA was stained using SYBR® _{Green I} Nucleic Acid Gel Stain. DNA samples were mixed with 10 × Orange G loading dye before being loaded onto the gel along with 1 Kb Plus DNA Ladder standard (Invitrogen). 1 L of 1× TAE buffer was used as a running buffer. Electrophoresis was carried out at 80 V for 50 min at ambient

temperature using Electrophoresis Subsystem 150 apparatus (LabNet). Bands were visualized by UV Transilluminator at 254 nm, and imaged using GLEDOC software.

2.2.3.7 Phylogenetic analysis TE buffer

10 mM Tris-HCl and 1mM EDTA were mixed in distilled water. 2M NaOH was used to adjust the pH of the solution to 8.0. Solution was sterilized by autoclaving.

PCR amplification

PCR amplification of 16S rRNA gene of strain 14T _{was performed using universal primers 8F (5’-} AGAGTTTGATCCTGGCTCAG-3’) and 1510R (5’-GGTTACCTTGTTACGACTT-3’). 25 mM of lyophilised primers were purchased from Life Technologies NZ Ltd. Primers were reconstituted in TE buffer to make up 100 µM primer stock solutions. HotStarTaq® _{Master Mix Kit was purchased from} QIAGEN. A 50 µl PCR reaction contained 25 µl of HotStarTaq® _{Master Mix (contains 2.5 units of} HotStarTaq® _{DNA polymerase; 1× MgCl}

µM forward primer; 10 µl of 2 µM reverse primer; 4 µl RNase-free water; and 1 µl of template DNA. Optimized PCR cycling conditions recommended by the manufacturer were used: initial heat

polymerase activation at 95℃ for 15 min, 35 cycles of 95℃ for 30 sec (denaturation), 56℃ for 30 sec (annealing), 72℃ for 90 sec (extension), final extension at 72℃ for 10 min, and storage at 4℃ until further processing. PCR was performed using Applied Biosystems GeneAmp® _{PCR System 9700.} PCR products were cleaned using QIAquick® _{PCR Purification Kit (QIAGEN) according to the} manufacturer’s instruction. The purity and quality of DNA samples were checked by Nanodrop®_-1000 measurement and gel electrophoresis. DNA quantification was carried out using Qubit™ dsDNA BR assay.

Vector ligation and transformation of competent cells

Purified PCR products were ligated into pGEM®_{-T Easy Vector (Promega) using 1:5 vector: insert} ratio. A standard ligation reaction contained 10 µl 2× Rapid Ligation Buffer; 1 µl of pGEM®_{-T Easy} Vector (50 ng); 5 µl of PCR product (~250 ng); 2 µl of T4 DNA ligase (3 Weiss units/µl); and 2 µl of sterile water. After mixing by gentle pipetting, the reactions were incubated at 4℃ overnight. 3 µl of ligation reaction was added into a vial of One Shot® _{TOP10 Chemically Competent E.coli}

(Invitrogen) and mixed by gentle tapping. The vials were incubated on ice for 30 min and then heat- shocked for 30 sec at 42℃ in a water bath. Vials were immediately transferred on ice and incubated for further 2 min. 250 µl of pre-warmed S.O.C medium (Invitrogen) was added into each vial of competent cells. Vials were shaken horizontally at 37℃ for 1 h at 225 rpm. 50 µl of transformation mixture was spread on pre-warmed LB agar plates containing 100 µg/ml ampicillin and 1.6 mg X-gal per plate. Plates were incubated at 37℃ overnight. White colonies were selected and grown in 5 ml LB broth medium containing 100 µg/ml ampicillin overnight. Cells were collected for plasmid extraction by centrifuging at 10,000 g for 5 min at room temperature.

Isolation of plasmid DNA

Plasmid DNA was extracted from transformed cells using PureLink® _{Quick Plasmid Miniprep Kits} (K2100-10, Invitrogen) using the manufacturer’s instruction.

DNA sequencing

Sequencing of recombinant plasmids was carried out at Genome Service Centre (Massey University, New Zealand) using BigDye® _{Terminator v3.1 or v3.0 Cycle Sequencing Kit on ABI3730 capillary} instrumentation (Applied Biosystems). Each sequencing reaction contained 100-300 ng of DNA; 3.2 pmole primer; 1× BigDye® Terminator chemistry; 5× sequencing reaction buffer; and sterile water to make up the final volume to 20 µl. M13 forward (5’-CCCAGTCACGACGTTGTAAAACG-3’) and M13 reverse (5’-AGCGGATAACAATTTCACACAGG-3’) primers were supplied by the sequencing service provider. Sequencing results were provided in ABI format.

Phylogenetic analysis

Multiple sequence alignments were performed on ClustalW using default parameters using Mega7 software (Kumar et al., 2016). Phylogenetic tree was constructed using neighbor-joining method (Saitou and Nei, 1987). Bootstrap values were calculated using 2,000 re-sampling to evaluate the support of tree topology. Reference 16S rRNA gene sequences from type strains and cloned 16S rRNA gene sequences from uncultured bacteria were obtained from GenBank database. 16S rRNA gene sequence from Lutispora thermophila strain EBR46T (GenBank accession number NR_041236) was used as outgroup.

2.2.3.8 G+C mol % analysis of DNA

The G+C content of DNA was determined as a part of whole-genome sequencing process using Illumina HiSeq 2500 (Macrogen, Korea).

2.2.3.9 Cellular fatty acid analysis by GC

Cellular fatty acid contents were determined by gas chromatography by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunscheweig, Germany).

2.2.3.10 Oxygen sensitivity

Strain 14T _{was subcultured 5 times under aerobic and microaerophilic (2 % or 4 % O}

2 inside Thermo™ Scientific CO2 incubator) conditions. Cell viability after 1 week of each transfer was observed using Olympus CX21 microscope.

2.2.3.11 Optimum temperature

Strain 14T _{was inoculated in mineral medium containing 0.5 % (w/v) citrus pectin. Cultures were} incubated at a range of temperatures (25-45℃ at 5℃ intervals). Optical density at 595 nm was measured daily on Spectronic 20 spectrophotometer (Bausch & Lomb) until stationary phase was reached.

2.2.3.12 Optimum pH

Growth of strain 14T over a pH range of 5.0-9.0 (0.5 intervals) at 36℃ was monitored. Using 2M NaOH and 2M HCl, pH of RC medium was adjusted before autoclaving. RC medium was used instead of mineral medium as it was difficult to manipulate pH in a CO2-gased medium. Prior to inoculation, 0.5 % (w/v) citrus pectin was added to 9 ml sterile medium. Changes in optical density at 595 nm were measured daily on Spectronic 20 spectrophotometer (Bausch & Lomb) until stationary phase was reached.

2.2.3.13 Substrate utilization Carbohydrate Solutions

Starch from wheat, arabinan from sugar beet (Megazyme), xylan from oat spelt, galactan from ex gum arabic (Sigma-Aldrich), β-glucan from barley, arabinoxylan from wheat (Megazyme), and

oligofructose-enriched inulin from chicory (Orafti® Synergy1) were weighed and dispensed into anaerobic water to make up 5 % (w/v) solutions. Substrates were dissolved by heating in a water bath at 65℃ for 20 min. Solubilized substrates were filtered (0.45 µm; Merck Millipore) into N2-washed sterile Hungate tubes. Substrate solutions were added into mineral medium at a final concentration of 0.5 % (w/v) prior to inoculation.

Sugar Solution

D-glucose, D-mannitol, D-lactose, D-sucrose, D-maltose, salicin, D-xylose, L-arabinose, cellobiose, D-mannose, D-sorbitol, L-rhamnose, D-galacturonic acid, D-raffinose, D-galactose, D-fructose, D- galactitol, and D-fucose were weighed and dissolved in anaerobic water to make up 5 % (w/v) sugar solutions. Solutions were filter-sterilized using 0.22 µm filters (Merck Millipore) into N2-washed sterile Hungate tubes. Prepared solutions were injected into mineral medium at a final concentration

of 0.5 % (w/v) prior to inoculation. 5 % (v/v) glycerol solution was boiled and cooled on ice while bubbling with N2. 0.05 % (w/v) L-cysteine-HCl was added before dispensing the solution into Hungate tubes. Solution was sterilized by autoclaving. Glycerol solution was added to mineral medium at a final concentration of 0.5 % (v/v).

Insoluble Substrates

Whatman filter papers (number 1) were cut into 5 × 5 mm squares. 10 pieces were added into an empty Hungate tube which was then topped with 9 ml of freshly prepared mineral medium dispensed under CO2. 50 mg of Avicel® PH-101 (~50 µm particle size; Fluka Analytical) and Sigmacell

cellulose (Type 101) were weighed and dispensed into empty Hungate tubes. 9 ml of freshly prepared mineral medium was dispensed into the tubes under CO2. Sealed medium containing filter papers and cellulose substrates was sterilized by autoclaving.

Growth experiments

The growth of strain 14T _{on substrates mentioned above was examined by monitoring the culture} viability over five successive transfers. The changes in optical density at 595 nm was measured daily on FLUOstar Optima Microplate Reader (BMG Labtech). Gram stained cells were observed under a light microscope to observe the growth of cells.

2.2.3.14 Size exclusion chromatography (SEC) Sample preparation

100 µl of clarified rumen fluid, 100 µl of 5 % (w/v) apple or citrus pectin solutions, and 100 µl of 1- week-old inoculum were added in triplicate to 1.7 ml mineral medium. Cultures were grown at 37℃ for 3 days with a constant shaking. 900 µl of samples were taken out at 48 h and 72 h of incubation. These samples were centrifuged at 12,000 g for 10 min at room temperature. Supernatants were transferred to clean Eppendorf tubes and sent to Ferrier Research Institute (Victoria University of Wellington) for SEC analysis.

SEC analysis

Polysaccharide substrates were dissolved in 0.1 M NaNO3 (2 mg/mL), allowed to hydrate fully by standing at room temperature overnight and centrifuged (14,000 g, 10 min) to clarify. The soluble material and samples of spent culture media (100 µL) were injected and eluted with 0.1 M NaNO3 (0.5 mL/min, 60°C) from three columns (TSK-Gel G5000PWXL, G4000PWXL and G3000PWXL, 300x 7.8 mm, Tosoh Corp., Tokyo, Japan) connected in series. The eluted material was detected using a refractive index monitor. The system was also calibrated with a series of pullulan molecular weight standards (6–850 kDa; Shodex, Showa Denko K.K. Tokyo, Japan).

Spent culture media (100 µL) were also injected and eluted with 0.1 M NaNO3 (0.5 mL/min, 60°C) from two Superdex Peptide (GE Healthcare) columns in series. The eluted material was detected