Characterization of Bifidobacterium kashiwanohense that utilizes both milk- and plant-derived oligosaccharides

Figures & data

Table 1. Bifidobacterium kashiwanohense associated strain list^{26,27,30–37.}

Strain	Isolation	Contig	Genome Size (Mbp)	No. of CDSs	Accession Number	Reference
B. kashiwanohense JCM 15,439^T	Japanese infant feces	1	2.34	1,956	GCA_001042615	Morita et al.(2015)
B. kashiwanohense APCKJ1	Irish infant feces	1	2.45	2,099	GCA_009684555	James et al. (2019)
B. kashiwanohense YIT 13,051	Japanese child feces	28	2.35	2,024	JAOPLX000000000	This study
B. kashiwanohense YIT 13,052	Japanese child feces	16	2.33	1,962	JAOPLY000000000	This study
B. kashiwanohense YIT 13,053	Japanese child feces	15	2.34	1,975	JAOPLZ000000000	This study
B. kashiwanohense YIT 13,054	Japanese child feces	23	2.26	1,898	JAOPMA000000000	This study
B. kashiwanohense YIT 13,055	Japanese child feces	19	2.37	1,989	JAOPMB000000000	This study
B. kashiwanohense YIT 13,056	Japanese child feces	18	2.28	1,904	JAOPMC000000000	This study
B. kashiwanohense YIT 13,057	Japanese infant feces	56	2.52	2,155	JAOPMD000000000	This study
B. kashiwanohense YIT 13,058	Japanese infant feces	30	2.43	2,131	JAOPME000000000	This study
B. kashiwanohense YIT 13,060	Belgian infant feces	31	2.37	2,015	JAOPMF000000000	This study
B. kashiwanohense YIT 13,061	Belgian infant feces	17	2.3	1,958	JAOPMG000000000	This study
B. kashiwanohense YIT 13,062	Belgian infant feces	32	2.53	2,249	JAOPMH000000000	This study
B. catenulatum JCM 1194^T	Human feces	1	2.08	1,702	GCA_001025195	Morita et al. (2015)
B. catenulatum 1899B	Infant feces	14	2.12	1,758	GCA_002075855	Duranti et al. (2017)
B. catenulatum BIOML-A1	Human feces	18	2.06	1,737	GCA_009160805	Poyet et al. (2019)
B. catenulatum YIT 13,059	Japanese infant feces	19	2.03	1,622	JAOPMI000000000	This study
[B. kashiwanohense] PV20–2	Kenyan infant feces	1	2.37	1,991	GCA_000800455	Vazquez-Gutierrez et al. (2015)
[B. kashiwanohense] N4G05	Kenyan vaginal fluid	12	2.07	1,678	GCF_002742445	Freitas & Hill (2017)
[B. kashiwanohense] N5G01	Kenyan vaginal fluid	8	2.12	1,714	GCF_002742425	Freitas & Hill (2017)
[B. catenulatum] GCF_901212515	Bangladeshi children feces	28	2.23	1,842	GCA_901212515	Cowardin et al. (2019)
[B. catenulatum] GCF_902167655	Bangladeshi children feces	36	2.17	1,802	GCA_902167655	Raman et al. (2019)
[B. catenulatum] GCF_902167905	Bangladeshi children feces	75	2.18	1,853	GCA_902167905	Gehrig et al. (2019)

Figure 1. General genome features of B. kashiwanohense.

(a) Phylogenic tree of B. kashiwanohense-associated strains based on their core genome single nucleotide polymorphisms (SNPs). (b) Number of coding sequences (CDSs) in human bifidobacterial species. (c) Pan-genome and core-genome of B. kashiwanohense. (d) Venn diagram of core genes of B. kashiwanohense and B. catenulatum. (e) Distribution of B. kashiwanohense on each continent. The size of the pie chart represents the number of metagenomes used for estimating the distribution of this subspecies. (f) Abundance of the Bifidobacteriales, including B. kashiwanohense, during the first 2 years of life¹⁴. White and black arrows represent the initiation of solid food and cessation of breastfeeding, respectively.

Figure 2. B. kashiwanohense glycobiome and associated growth profiles.

Note: (a) Glycosyl hydrolase (GH) family gene profiles of 13 B. kashiwanohense strains in comparison with other bifidobacterial strains (n = 350 genomes in total). The strains (y-axis) and GH family scale abundance (x-axis) have been hierarchically clustered by measuring the Euclidean distance with complete linkage clustering. A black box in the figure represents the GH profiles of B. kashiwanohense. The red and green boxes represent the HMO utilization and plant-derived carbohydrates utilization GH genes, respectively, that are harbored by B. kashiwanohense strains. The pink box represents B. bifidum specific GH genes. (b) Principal component analysis (PCA) of the GH profile of each strain. (c) Explained variance plot. Red and green represent featured GH genes associated with HMO and plant-derived carbohydrate metabolism, respectively. (d) Growth of B. kashiwanohense and other Bifidobacterium strains with different carbohydrates as substrates. The growth of each strain was determined as optical density (OD₆₀₀) at the end of cultivation (60 h), and the result is visualized in the heatmap.

Figure 3. Utilization of xylan-associated carbohydrates by B. kashiwanohense.

Note: (a) Growth curves of 12 B. kashiwanohense strains cultured with xylooligosaccharide (XOS), arabinoxylan (AX), and xylan as substrates. (b) Organization of genes for xylan-related carbohydrate utilization (right) and the associated phenotype (left). The shadows (grayscale) represent the homology among strains. (c) Proposed metabolic pathway of AX conversion to monosaccharides. The glycosidases and transporters involved in xylan-related carbohydrate utilization are colored as in Figure 3b.

Figure 4. HMO utilization by B. kashiwanohense.

Note: (a) Growth curves of 12 B. kashiwanohense strains cultured in the presence of 2′-FL, 3-FL, and HMO mixture. (b) HPLC profiles of the HMOs remaining in the culture supernatants after 72 h. (c) Organization of genes for HMO utilization and the associated phenotype. A difference in ABC transporter SBP subtypes between LNDFH-utilizing and non-utilizing strains was noted. (d) Model of HMO utilization by B. kashiwanohense strains. The glycosidases and transporters involved in HMO utilization are colored as in Figure 4d.

Figure 5. B. kashiwanohense strains utilize both milk- and plant-derived carbohydrates.

Note: B. kashiwanohense strains possess unique SBP that transports not only short- but also long-chain HMOs. Most B. kashiwanohense strains possess an extracellular xylanase homolog that suggests it enables primary degradation of arabinoxylan, ABC transporters for xylan-based oligosaccharides, and an intercellular xylosidase. This carbohydrate utilizing strategy is unique and contrasts that of infant-associated bifidobacteria (e.g., B. infantis and B. breve) utilizing HMOs and adult-associated species (e.g., B. pseudocatenulatum) utilizing dietary fiber.

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Data availability statement

The bifidobacterial genome sequences have been deposited in the NCBI Sequence Read Archive under BioProject Accession Code PRJNA883016 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA883016)