Genome sequencing
We sequenced the genomes of 35 strains of B. pseudocatenulatum (Supplementary Table S1). These strains were isolated at the Yakult Central Institute and the species were identified based on the 16S rRNA gene sequence analysis. These strains have been isolated in the course of various studies over the past few decades, including many studies on infants and adults. B. pseudocatenulatum cultures were anaerobically incubated in modified Gifu anaerobic medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with lactose and glucose (both 0.5% wt/vol) at 37 °C for 16 h. These culture conditions were applied throughout the study unless stated otherwise. The detailed procedures for genomic DNA extraction, library preparation for MiSeq (Illumina, San Diego, CA, USA), MinION (Oxford Nanopore Technologies, Oxford, UK) and PacBio RS2 (Pacific Biosciences, Menlo Park, CA, USA), and sequencing are described in the Supplementary Methods.
Genome assembly, gene prediction and pangenome analysis
We used Unicycler [26] with default parameters for both short-read and hybrid assembly, and Prokka [27] with default parameters for annotating the reconstructed genomes and those downloaded from the RefSeq database. The annotated genomes were then processed with Roary [28] with a default gene identity cut-off parameter of 95% for species level pangenome analysis. A representative sequence from each gene cluster was translated into a protein sequence, and CAZymes were identified using the dbCAN2 server [29]. Proteins were considered CAZymes if they were identified using HMMER, DIAMOND and Hotpep with default parameters. We then built a CAZyme gene distribution matrix (Supplementary Table S2) based on the gene presence-absence table determined using Roary.
Carbohydrate utilisation assays
Strains of B. pseudocatenulatum were cultured until they reached the exponential phase, centrifuged, and then, the resulting pellets were suspended to an OD600 of 0.2 in modified peptone yeast extract (PY) medium (100 mM PIPES, pH 6.7, 2 g/L peptone, 2 g/L BBL trypticase peptone, 2 g/L bacto-yeast extract, 8 mg/L CaCl2, 19.2 mg/L MgSO4 ∙ 7H2O, 80 mg/L NaCl, 4.9 mg/L hemin, 0.5 g/L L-cysteine hydrochloride and 100 ng/L vitamin K1). These suspension cultures were inoculated (1% vol/vol) into modified PY medium supplemented with 0.5% (wt/vol) XOS (Xylo-Oligo95P, B Food Science, Aichi, Japan) (PY-XOS), wheat arabinoxylan (Megazyme, Bray, Ireland) (PY-AX) or beechwood xylan (Sigma-Aldrich, Darmstadt, Germany) (PY-XY) and covered with sterile mineral oil (50 μL) to prevent evaporation. Growth was monitored anaerobically by measuring the OD600 using a PowerWave 340 plate reader (BioTek, Winooski, VT, USA) every 30 min in an anaerobic chamber for 48 h. The organic acids produced in PY-XY were analysed using high-pressure liquid chromatography as described [8].
Cloning, expression and purification of recombinant BpXyn10A
The GH10 domain of the BpXyn10A gene was amplified by PCR using the primers xynA-GH-F (5’-CATCATCATCATCATGCGGAAGGCGACGCCGTA-3’) and xynA-GH-R (5’-AGCAGAGATTACCTAATCCTTGAATGCGTTCATGC-3’), with the genomic DNA of YIT 11027 as a template. A linearised vector was synthesised by PCR using primers pColdII-F (5’-GTAATCTCTGCTTAAAAGCACAGAATCTA-3’) and pColdII-R (5’-ATGATGATGATGATGATGCACTTTGT-3’), and the pColdII vector (Takara Bio, Otsu, Japan) as a template. These fragments were ligated using In-Fusion HD Cloning Kits (Takara Bio, Otsu, Japan), resulting in pColdII-xynA. Escherichia coli BL21 was transformed with pColdII-xynA and cultured to express recombinant BpXyn10A as described by the manufacturer. Bacterial cells were harvested by centrifugation and lysed with B-PER Bacterial Cell Lysis Reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing lysozyme at 100 µg/mL and 10 U/mL of DNase I. Recombinant BpXyn10A was further purified using Ni-NTA Spin Column (Qiagen, Hilden, Germany) and analysed by SDS-PAGE.
Endo-xylanase activity assay
B. pseudocatenulatum YIT 11027, YIT 11952 and YIT 4072T cells were grown anaerobically in PY-AX or PY-XOS medium for 16 h. Cultures (1.5 mL) were centrifuged (8000× g for 2 min at room temperature); then, supernatants were sterilised by passage through a 0.22-μm filter. Pelleted cells were washed with modified PY medium and resuspended in 1.5 mL of the same medium. The endo-xylanase activity of the supernatant and the cell fractions were assayed using Xylanase Assay kits (XylX6 method) (Megazyme, Bray, Ireland) as described by the manufacturer. According to the manufacturer, this kit is designed to specifically detect only endo-xylanase activity, and not xylosidase or exo-xylanase enzyme activity.
Purified BpXyn10A-added culture
B. pseudocatenulatum YIT 4072T and Ba. ovatus YIT 6161T cells were cultured anaerobically until they reached the exponential phase. Thereafter, cultures (200 μL) were centrifuged (8000× g for 2 min at room temperature), then pelleted cells were resuspended in modified PY medium (500 μL), and inoculated (1% vol/vol) into PY-AX medium supplemented with 0, 10, 100 and 1000 ng/mL purified recombinant BpXyn10A. Growth was monitored anaerobically by measuring the OD600 using the PowerWave 340 plate reader.
RNA-seq analysis
B. pseudocatenulatum YIT 11952 was cultured in modified PY medium supplemented with 0.5% (wt/vol) lactose, xylose, XOS, beechwood xylan or arabinoxylan and harvested at mid- to late-log phase. The detailed procedures for total RNA extraction, rRNA removal and sequencing using MiSeq are described in the Supplementary Methods. We obtained a total of 23 million paired-end reads. Low-quality bases (average quality <30) were trimmed off at the 3’, and the resulting reads with N bases, or <70 bp long, were filtered out using cutadapt [30]. Ribosomal RNA reads were removed using SortMeRNA [31]. Filtered reads were then mapped to the complete genome sequence of B. pseudocatenulatum YIT 11952 using Bowtie2 [32], and read counts of each gene were determined with featureCounts [33]. The expression levels of each gene were quantified as transcripts per million calculated using Microsoft Excel 2013.
Batch co-cultures of B. pseudocatenulatum and Ba. ovatus
The 36 strains of B. pseudocatenulatum and Ba. ovatus YIT 6161T were separately grown before co-culture. Cells harvested at the mid- to late-log phase were resuspended in modified PY medium adjusted to an OD600 of 0.2 (B. pseudocatenulatum strains; equivalent to 1 × 108.4–9.0 cells/mL) or 0.02 (Ba. ovatus, equivalent to 1 × 108.7–9.0 cells/mL) so that the numbers of cells were similar. Equal amounts of each suspension were inoculated at 2% (vol/vol) into the PY-AX medium. After 48 h of anaerobic co-culture, DNA was extracted using the beads-phenol method as described above. Each sample was analysed by quantitative PCR using an AB7500 real-time qPCR system (Thermo Fisher Scientific, Waltham, MA, USA) to determine cell numbers with specific primers for B. pseudocatenulatum (BiCATg-1: 5’-CGGATGCTCCGACTCCT-3’ and BiCATg-2: 5’-CGAAGGCTTGCTCCCGAT-3’) [10] and Ba. ovatus (g-Bfra-f: 5’-ATAGCCTTTCGAAAGRAAGAT-3’ and g-Bfra-r: 5’-CCAGTATCAACTGCAATTTTA-3’) [34], respectively.
Co-culture time-series experiment between B. pseudocatenulatum and B. longum subsp. longum
B. pseudocatenulatum YIT 11952 and B. longum subsp. longum H11-1 (isolated from an infant) were separately grown before co-culture. Cells harvested at the mid- to late-log phase were resuspended in modified PY medium adjusted to an OD600 of 0.2. Equal amounts (2% vol/vol) of each suspension were inoculated into 500 μL of PY-AX medium. After 0, 8, 24 and 48 h of anaerobic co-culture, DNA was extracted from subsamples (50 μL) for quantitative real-time PCR using specific primers for B. pseudocatenulatum (BiCATg-1 and BiCATg-2) and B. longum subsp. longum (BiLON-1: 5’-TTCCAGTTGATCGCATGGTC-3’ and BiLON-2: 5’-GGGAAGCCGTATCTCTACGA-3’) [10] using the ABI PRISM 7500 PCR system to determine cell numbers.
Cereal intervention
Experimental design
This study was approved by the ethical committee of the Yakult Central Institute, in accordance with the committee guidelines and the Declaration of Helsinki (2013). This study is registered at UMIN Clinical Trials Registry (number UMIN 000043680). Written informed consent was obtained from 30 Japanese adult participants to participate in this study. Three participants who were prescribed with medications during the experimental period were excluded from data analysis. The experiment was completed by 27 (19 males and 8 females) participants aged 28–65 years who consumed 30 g of All Bran Original wheat bran-rich cereal (Kellogg Japan, Tokyo, Japan) with 180 mL of Accadi lactose-digested milk (Megmilk Snow Brand, Tokyo, Japan) once in the morning and once in the afternoon daily. According to the manufacturer, 60 g of cereal provided approximately 6.6 g of wheat bran arabinoxylan per day. Because almost the entire Japanese population has the genotype for low lactase activity [35], we used 80% lactose-depleted milk according to the manufacturer to minimise the effect of lactose on the growth of Bifidobacterium independently of cereal consumption. The experiment comprised pre-intervention (days 1–7), intervention (days 8–14) and post-intervention (days 15–21) periods. Throughout the 21 days of the experiment, the participants were instructed to refrain from consuming fermented milk containing bifidobacteria, prebiotic products and foods rich in LCX, such as other bran-rich cereal, whole grain or brown rice.
Faecal samples were collected on days 4, 7, 11, 14, 18 and 21. To reduce daily individual variations, we averaged the results of individual samples from each period. On days without defecation, faecal samples were collected the next day. If no defecation occurred on days 7, 14 or 21, the previous period was extended until a faecal sample was collected. None of the participants skipped the regime for more than 2 days. Faecal samples collected immediately after defecation were placed in sterile tubes, kept on ice and then stored at −80 °C. Thereafter, DNA was extracted from ten-fold diluted faecal samples using the beads-phenol method as described above.
Quantitation of B. pseudocatenulatum in faecal samples
B. pseudocatenulatum cells were quantified using real-time PCR with a BiCATg primer that targets the 16S rRNA gene. This primer set has been confirmed to be specific for B. pseudocatenulatum and B. catenulatum and it does not react non-specifically with other bifidobacteria [10]. Although this primer set detects both B. pseudocatenulatum and B. catenulatum [10], we regarded the measured value as that of only B. pseudocatenulatum because none of the participants had amplicon sequence variants (ASVs) close to B. catenulatum in the 16S rRNA gene amplicon analysis described below. The PCR mixture (20 μL total volume) contained 1 × TB Green Premix Ex Taq II (Takara Bio, Otsu, Japan), 0.2 μM of each primer and 2 μL of template DNA. The thermocycling conditions used were 94 °C for 10 s, followed by 40 cycles of 94 °C for 20 s, 55 °C for 20 s and 72 °C for 50 s, then a melting-curve programme. Amplification was performed using the ABI PRISM 7500 Real-Time PCR System. For absolute quantification, a standard curve was calculated using a ten-fold dilution series of DNA extracted from the B. pseudocatenulatum strain with BpXyn10A, for which cell numbers were determined. The detection limit for this system was 5.0 × 104 cells/g faeces.
Quantification of cells with BpXyn10A in faecal samples
The numbers of cells with BpXyn10A were quantified by real-time PCR with BpXyn10A gene-targeted oligonucleotide primers. To design specific primers, we used the sequences of all BpXyn10A genes and their homologues obtained from an NCBI blastn search, aligned them, and detected the specific region of the BpXyn10A gene. Then, we designed pBpXyn10A-F (5’-CGAGAATGCGAACACGTACTTC-3’) and pBpXyn10A-R (5’-CTGCTCGGTGTTGTAATCGTTG-3’), which provided a 94 bp amplicon. Using Primer-BLAST [36], we confirmed that there were no non-specific sequences that could be amplified in the sequences registered in the NCBI nr database. Furthermore, we performed PCR using DNA from the B. pseudocatenulatum 35 strains used in our study, and confirmed that specific amplification products were obtained only from strains with the BpXyn10A gene. The PCR mixture (20 μL total volume) contained 1 × TB Green Premix Ex Taq II (Takara Bio, Otsu, Japan), 0.4 μM of each primer and 2 μL of template DNA. The thermocycling conditions used were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s, and a melting-curve programme. Amplification was performed using the ABI PRISM 7500 Real-Time PCR System.
16S rRNA gene amplicon analysis
The V1–2 regions of the 16S rRNA gene were amplified. Primer sequence, PCR conditions and details on library preparation are described in the Supplementary Methods. Pooled amplicons were sequenced using the MiSeq platform with the MiSeq reagent kit v2 500 cycle. Obtained reads were analysed using the QIIME2-2020.8 platform [37]. Briefly, the sequence data were denoised using DADA2, and the resulting ASVs were assigned taxonomy in QIIME2 using the SILVA138 database. Alpha-diversity (observed_features, faith_pd and Shannon_index) and beta-diversity (unweighted UniFrac) were analysed using QIIME2. Homology of the ASVs was also analysed using vsearch with the 16S RefSeq nucleotide sequence records to determine the closest species. To analyse species level trends, we grouped ASVs with >97% homology to closely related species as a single species. No ASVs were more closely related to B. catenulatum than to B. pseudocatenulatum.
Statistics
Data were statistically analysed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Average CAZyme gene copy numbers of each species in the genome, amounts of organic acids produced in PY-XY medium and numbers of cells with and without BpXyn10A in the co-culture experiments were compared using Mann–Whitney U tests. The effects of the presence or absence of Bacteroides species on the growth of B. pseudocatenulatum in co-culture experiments, and the increase or decrease of each bacterial species in the cereal intervention study were verified using Wilcoxon signed-rank tests.
Source: Ecology - nature.com
