ABSTRACT
The genome sequences of two Faecalibacterium strains isolated from a healthy Japanese individual were analyzed. Comparative genomics revealed the smallest genome within the genus to date and identified core gene clusters, providing new insights into the minimal genomic requirements and evolutionary adaptation of Faecalibacterium species.
KEYWORDS: Faecalibacterium, human gut microbiota, butyrate-producing bacteria
ANNOUNCEMENT
Faecalibacterium species are among the most abundant butyrate-producing bacteria in the human gut and play a key role in maintaining gut barrier integrity and modulating inflammation (1). Increasing evidence supports their health-promoting properties and highlights them as promising next-generation probiotics (2). However, genomic information on Faecalibacterium species other than Faecalibacterium prausnitzii remains limited, especially in Asian populations (3, 4). To address this gap and to better understand the diversity and functional potential of Faecalibacterium in the Japanese gut, we isolated two novel strains, F15 and a30, from a healthy individual and determined their draft genome sequences.
A fecal sample from a healthy 22-year-old Japanese man from Okayama, Japan, was serially diluted under anaerobic conditions and plated on modified GAM agar containing 1% pectin. After 48 hours of anaerobic incubation at 37°C, colonies with typical Faecalibacterium morphology were selected and identified via 16S rRNA sequencing using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1525R (5′-AAGGAGGTGATCCAGCC-3′) (Eurofins) on a 3730xl DNA Analyzer (Applied Biosystems) (5). The isolates were cultured in modified GAM broth with 0.5% sodium acetate at 37°C for 24 hours anaerobically. Cells were harvested, washed, and treated sequentially with lysozyme, achromopeptidase, proteinase K, and SDS. Genomic DNA was extracted using phenol:chloroform:isoamyl alcohol, purified by ethanol precipitation and centrifugation, treated with RNase, further purified using NaCl and PEG precipitation, washed with ethanol, and resuspended in TE buffer (6).
Genomic libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina) and sequenced with 300-base paired-end reads on the MiSeq platform using the MiSeq Reagent Kit v3 (Illumina). Sequencing reads were quality filtered to remove low-quality bases and short reads, and subsequently assembled de novo using CLC Genomics Workbench v10.1.1 with default parameters. Protein-coding genes were annotated using DFAST v1.6.0 (7). A summary of the sequencing and genome assembly statistics is presented in Table 1. For species-level classification of Faecalibacterium, the recA gene proved more reliable than the 16S rRNA gene as a phylogenetic marker (8). The recA sequence of strain F15 showed 99.1% identity to Faecalibacterium duncaniae JCM 31915T (9) and an average nucleotide identity (ANI) of 96.7%, supporting its classification as F. duncaniae (Fig. 1). Strain a30 showed 99.1% recA sequence identity to Faecalibacterium taiwanense HLW78T (10) and an ANI of 97.8%, consistent with classification as F. taiwanense (Fig. 1).
TABLE 1.
Genomic features of two Faecalibacterium strains analyzed in this study
| Species | Strain | No. of reads | No. of contigs | Genome size (bp) | N50 contig size (bp) | GC content (%) | Genome coverage (×) | No. of protein-coding genes |
|---|---|---|---|---|---|---|---|---|
| F. duncaniae | F15 | 550,916 | 141 | 2,988,843 | 44,918 | 56.2 | 55.3 | 2,791 |
| F. taiwanense | a30 | 481,940 | 89 | 2,554,504 | 55,053 | 56.6 | 56.6 | 2,308 |
Fig 1 .
Neighbor-joining phylogenetic tree based on the recA gene sequences showing the relationships between the two analyzed strains and related Faecalibacterium species. The recA gene sequences for each species were obtained from reference genomes available at NCBI. Multiple sequence alignment was performed using ClustalW2, and the phylogenetic tree was constructed using MEGA11 (11). Values in parentheses indicate genome size in megabases. The scale bar indicates the number of nucleotide substitutions per site.
Remarkably, strain a30 has the smallest genome (2.55 megabases) among all Faecalibacterium species reported to date. To estimate the core genome of the genus, we compared the gene clusters of strain a30 with those of 11 other reference strains representing Faecalibacterium. A total of 1,203 genes were shared by all 12 genomes and were inferred to constitute the core gene cluster of Faecalibacterium species. These findings provide new insights into the minimal genomic requirements for the genus and establish a foundation for understanding its essential functions and evolutionary adaptations.
ACKNOWLEDGMENTS
The authors thank Takehiro Matsubara (Okayama University Hospital Biobank) for providing Illumina sequencing services. The human fecal sample was obtained from a healthy Japanese man who provided informed consent. This study was approved by the Okayama University Ethics Committee (#1610-026). This work was supported by a JSPS KAKENHI Grant-in-Aid for Scientific Research (grant number JP23K18086). The authors note with sadness that Hidetoshi Morita, a co-author of this manuscript, passed away prior to its submission. His contributions are gratefully acknowledged.
Contributor Information
Hidehiro Toh, Email: toh@nig.ac.jp.
Kensuke Arakawa, Email: karakawa@okayama-u.ac.jp.
Vanja Klepac-Ceraj, Wellesley College, Wellesley, Massachusetts, USA.
DATA AVAILABILITY
This study is associated with the BioProject registered under accession number PRJDB15359. Draft genome sequences for the two strains (F15 and a30) have been submitted to the DDBJ/GenBank/EMBL databases under the accession numbers BSSS01000001–BSSS01000141 and BSSR01000001–BSSR01000089, respectively. The raw sequencing data are available in the Sequence Read Archive under accession number DRA020529.
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Associated Data
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Data Availability Statement
This study is associated with the BioProject registered under accession number PRJDB15359. Draft genome sequences for the two strains (F15 and a30) have been submitted to the DDBJ/GenBank/EMBL databases under the accession numbers BSSS01000001–BSSS01000141 and BSSR01000001–BSSR01000089, respectively. The raw sequencing data are available in the Sequence Read Archive under accession number DRA020529.