# Comparative Genomics Revealed Genetic Diversity and Species/Strain-Level Differences in Carbohydrate Metabolism of Three Probiotic Bifidobacterial Species

Authors: Toshitaka Odamaki; Ayako Horigome; Hirosuke Sugahara; Nanami Hashikura; Junichi Minami; Jin-zhong Xiao; Fumiaki Abe
Journal: International Journal of Genomics
 (2015)

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## Abstract

Strains ofBifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve.
Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB), whereas B. animalis is a non-HRB species, several of the differences in these species’ gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics.


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## Body

## 1. Background

The genusBifidobacterium has been classified into 48 taxa, including 39 species and nine subspecies [1], three of which (Bifidobacterium longum,Bifidobacterium breve, andBifidobacterium animalis) are commonly applied as bifidobacterial probiotics in the food industry. Many studies have been performed on the functionality of probiotic bifidobacteria in the host, and the properties and functionality of probiotics have generally been thought to be strain dependent [2, 3]. However, previous reports have indicated that certain characteristics, such as the ability to generate vitamins [4], organic acids [5], and antimicrobial components [6, 7] as well as tolerance to stress [8, 9], are species dependent, although there is a degree of variation among strains. In several cases, it is unclear whether certain characteristics are species common or strain specific.Recent work has provided genomic information for all bifidobacterial species/subspecies [1, 10]. However, because the number of available genomic sequences from each species was limited in these studies, it is difficult to distinguish whether a number of unique genes are species or strain specific.To further elucidate bifidobacterial genetic diversity and to evaluate species-level differences, we performed comparative genomic analyses of 49 strains belonging to three bifidobacterial species, orB. longum,B. breve, andB. animalis, which are commonly applied as probiotics in the food industry. The use of a large set of genome sequences based on a relatively large number of strains of each species allowed the identification of pan-genome structures and the elucidation of differences in core genome structures among these species. In the present report, given that bifidobacteria have been reported to utilise many unique metabolic pathways for sugar fermentation [11–19], additional analysis was focused on the carbohydrate transport/metabolism of each species/strain.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Cultivation Conditions

The bacterial strains used in this study and general information about these strains are listed in Table1. The bifidobacterial strains were obtained from stock cultures maintained in the Morinaga Culture Collection (MCC; Morinaga Milk Industry Co., Ltd., Zama, Japan) and the American Type Culture Collection (ATCC, VA). Each strain was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine·HCl (Kanto Chemical, Tokyo, Japan) at 37°C for 16 h under anaerobic conditions before DNA extraction. The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies.Table 1
General genome features ofBifidobacterium species included in this study.
Species
Genotype based on
Strain
Status
Chromosome
Plasmid
Contig
Length (kbp)
CDSs
Source
GenBank accession number
16SrRNA
Whole genes
B. longumssp. infantis
infantis
infantis
ATCC 15697
Complete
1
2,833
2,416
Human infant faeces
CP001095
B. longumssp. longum
infantis
infantis
JDM301
Complete
1
2,478
1,958
Human vagina
CP002010
B. longumssp. longum
longum
longum
BBMN68
Complete
1
2,266
1,804
Human elderly faeces
CP002286
B. longumssp. infantis
longum
longum
157F
Complete
1
2
2,409
1,999
Human infant faeces
AP010890, AP010891, and AP010892
B. longumssp. longum
longum
longum
JCM 1217
Complete
1
2,385
1,924
Human infant faeces
AP010888
B. longumssp. longum
longum
longum
KACC 91563
Complete
1
2
2,396
1,985
Human infant faeces
CP002794, CP002795, and CP002796
B. longum
longum
longum
NCC2705
Complete
1
1
2,260
1,728
Human infant faeces
AE014295 and AF540971
B. longum
longum
longum
DJO10A
Complete
1
2
2,390
2,000
Human adult faeces
CP000605, AF538868, and AF538869
B. longumssp. longum
longum
longum
F8
Complete
1
2,385
1,681
Human feces
FP929034
B. longumssp. longum
longum
longum
ATCC 55813
Unfinished
140
2,373
2,109
Human infant faeces
ACHI00000000
B. longumssp. longum
longum
longum
CCUG 52486
Unfinished
55
2,453
2,240
Human elderly faeces
ABQQ00000000
B. longumssp. longum
longum
longum
1-6B
Unfinished
171
2,686
2,425
Human children feces
AJTF00000000
B. longumssp. longum
longum
longum
2-2B
Unfinished
141
2,625
2,412
Human children feces
AJTJ00000000
B. longumssp. longum
longum
longum
35B
Unfinished
131
2,514
2,260
Human infant faeces
AJTI00000000
B. longumssp. longum
longum
longum
44B
Unfinished
62
2,559
2,262
Human infant faeces
AJTM00000000
B. breve
breve
breve
ACS-071-V-Sch8b
Complete
1
2,327
1,826
Human vagina
CP002743
B. breve
breve
breve
UCC2003
Complete
1
2,423
1,852
Human infant faeces
CP000303
B. breve
breve
breve
DSM 20213
Unfinished
117
2,298
2,263
Human infant faeces
ACCG00000000
B. breve
breve
breve
CECT 7623
Unfinished
34
2,314
1,725
Human milk
AFVV00000000
B. breve
breve
breve
MCC0121
Unfinished
27
2,436
2,079
Human infant faeces
AVQA00000000
B. breve
breve
breve
MCC0305
Unfinished
22
2,287
1,927
Human adult faeces
AWFR00000000
B. breve
breve
breve
MCC0476
Unfinished
15
2,234
1,851
Human adult faeces
AVQB00000000
B. breve
breve
breve
MCC1094
Unfinished
23
2,327
1,944
Human infant faeces
AWFS00000000
B. breve
breve
breve
MCC1114
Unfinished
33
2,487
2,136
Human infant faeces
AVQC00000000
B. breve
breve
breve
MCC1128
Unfinished
25
2,480
2,143
Human infant faeces
AVQD00000000
B. breve
breve
breve
MCC1340
Unfinished
23
2,373
1,986
Human infant faeces
AWFT00000000
B. breve
breve
breve
MCC1454
Unfinished
14
2,457
2,111
Human infant faeces
AWFU00000000
B. breve
breve
breve
MCC1604
Unfinished
15
2,206
1,830
Human elderly faeces
AVQE00000000
B. breve
breve
breve
MCC1605
Unfinished
37
2,324
1,976
Human elderly faeces
AWFV00000000
B. animalisssp. animalis
animalis
animalis
ATCC 25527
Complete
1
1,933
1,538
Rat feces
CP002567
B. animalisssp. animalis
animalis
animalis
ATCC 27672
Unfinished
9
1,990
1,532
Rat feces
AWFQ00000000
B. animalisssp. animalis
animalis
animalis
MCC0483
Unfinished
27
2,176
1,750
Rat feces
AWFK00000000
B. animalisssp. animalis
animalis
animalis
MCC0499
Unfinished
11
2,134
1,721
Rat feces
AWFN00000000
B. animalisssp. animalis
animalis
animalis
MCC1489
Unfinished
14
1,910
1,474
Guinea pig feces
AWFO00000000
B. animalisssp. lactis
lactis
lactis
AD011
Complete
1
1,934
1,527
Human infant faeces
CP001213
B. animalisssp. animalis
lactis
lactis
ATCC 27536
Unfinished
18
1,912
1,561
Chicken feces
AWFL00000000
B. animalisssp. animalis
lactis
lactis
ATCC 27673
Unfinished
21
1,937
1,576
Sewage
AWFP00000000
B. animalisssp. animalis
lactis
lactis
ATCC 27674
Unfinished
18
1,912
1,561
Rabbit feces
AWFM00000000
B. animalisssp. lactis
lactis
lactis
B420
Complete
1
1
1,939
1,561
No information
CP003497
B. animalisssp. lactis
lactis
lactis
BB-12
Complete
1
1,942
1,642
Yoghurt
CP001853
B. animalisssp. lactis
lactis
lactis
Bi-07
Complete
1
1,939
1,597
No information
CP003498
B. animalisssp. lactis
lactis
lactis
Bl-04
Complete
1
1,939
1,567
Human infant faeces
CP001515
B. animalisssp. lactis
lactis
lactis
Bl12
Complete
1
1,938
1,518
Colonoscopic sample
CP004053
B. animalisssp. lactis
lactis
lactis
BLC1
Complete
1
1,944
1,518
No information
CP003039
B. animalisssp. lactis
lactis
lactis
BS01
Unfinished
7
1,932
1,572
No information
AHGW00000000
B. animalisssp. lactis
lactis
lactis
CNCM I-2494
Complete
1
1,943
1,660
No information
CP002915
B. animalisssp. lactis
lactis
lactis
DSM 10140
Complete
1
1,938
1,565
Yoghurt
CP001606
B. animalisssp. lactis
lactis
lactis
HN019
Unfinished
28
1,916
1,578
No information
ABOT00000000
B. animalisssp. lactis
lactis
lactis
V9
Complete
1
1,944
1,572
Human infant faeces
CP001892


### 2.2. Assimilation of Human Milk Oligosaccharides

Modified MRS was prepared by removing glucose from the original components and was supplemented with LNT (Dextra Laboratories Ltd., Reading, UK) at a final concentration of 2%. An aliquot of seed culture from each bifidobacterial strain was then inoculated into 200μL of the modified MRS, with a final concentration of 1%. Growth of each bifidobacterial strain was measured by absorbance at OD600 after cultivation under anaerobic conditions at 37°C for 24 h and 48 h. Experiments were performed in triplicate.


### 2.3. Genome Sequencing and Bioinformatics Analyses

Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The preparation of genomic libraries was performed with 1 ng of genomic DNA using the Nextera XT DNA Sample Preparation Kit (Illumina Inc., CA) according to the manufacturer’s instructions. After PCR amplification and cleanup, the fragment size distribution of the tagmented DNA was analysed using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA). The libraries were sequenced using a MiSeq Personal Sequencing System and the MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc.). Quality trimming andde novo assembly of the raw paired-end reads were performed using the CLC Genomics Workbench (v 6.0) software package (CLC bio, Aarhus, Denmark) with default settings, except for contig length (minimum contig length = 2,000 bp). Seventeen genomes that were sequencedde novo in this study were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2.0 and were manually checked as part of the process of genome submission to GenBank. Our sequencing efforts resulted in multiple contigs (Table 1). The datasets for the genome annotations for the other 32 strains published previously were retrieved from the FTP server of NCBI [20].


### 2.4. Pan-Genome Analysis

For all of the 49 bifidobacterial strains included in this study, a pan-genome calculation was performed using the PGAP [21]. The ORF content of each genome was organised into functional gene clusters using the gene family (GF) method. Under the GF method, the total protein sequences of each strain were mixed together, with each gene being marked as a strain identifier. BLASTALL searches were performed among the mixed protein sequences, and the filtered BLAST results were clustered using the Markov Cluster algorithm [22], which has been widely used in other studies on prokaryotic genomes and in programs designed to search for orthologues among multiple strains. For each gene pair in a given cluster, the global match region was no less than 50% of the longer gene protein sequence, and the identity was also no less than 50%. The minimum score value and E-value applied in BLAST were 50 and 1e-8. Pan-genome profile analysis, genetic variation analysis of functional genes, and function enrichment analysis of gene clusters were then performed. KEGG Orthology (KO) numbers were assigned by the KEGG Automatic Annotation Server (KAAS) using the bidirectional best-hit method [23].


### 2.5. Phylogenetic Trees

Pan-genome-based phylogenetic trees were generated according to a gene distance matrix, which was calculated based on genes that were absent or present in each strain [21], using UPGMA algorithms in PHYLIP. The sequences of 16S rRNA genes were aligned using the ClustalW alignment tool [24] with default parameters, and phylogenetic trees were constructed using UPGMA algorithms in MEGA6.06 [25]. Bootstrap values were calculated using 500 bootstrap replicates. The supertree was built using FigTree v1.4.0.


### 2.6. Nucleotide Sequence Accession Numbers

Thede novo sequence and annotation data reported herein have been deposited in GenBank under the accession numbers AVQA00000000-AVQE00000000 and AWFK00000000-AWFV00000000.


## 2.1. Bacterial Strains and Cultivation Conditions

The bacterial strains used in this study and general information about these strains are listed in Table1. The bifidobacterial strains were obtained from stock cultures maintained in the Morinaga Culture Collection (MCC; Morinaga Milk Industry Co., Ltd., Zama, Japan) and the American Type Culture Collection (ATCC, VA). Each strain was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine·HCl (Kanto Chemical, Tokyo, Japan) at 37°C for 16 h under anaerobic conditions before DNA extraction. The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies.Table 1
General genome features ofBifidobacterium species included in this study.
Species
Genotype based on
Strain
Status
Chromosome
Plasmid
Contig
Length (kbp)
CDSs
Source
GenBank accession number
16SrRNA
Whole genes
B. longumssp. infantis
infantis
infantis
ATCC 15697
Complete
1
2,833
2,416
Human infant faeces
CP001095
B. longumssp. longum
infantis
infantis
JDM301
Complete
1
2,478
1,958
Human vagina
CP002010
B. longumssp. longum
longum
longum
BBMN68
Complete
1
2,266
1,804
Human elderly faeces
CP002286
B. longumssp. infantis
longum
longum
157F
Complete
1
2
2,409
1,999
Human infant faeces
AP010890, AP010891, and AP010892
B. longumssp. longum
longum
longum
JCM 1217
Complete
1
2,385
1,924
Human infant faeces
AP010888
B. longumssp. longum
longum
longum
KACC 91563
Complete
1
2
2,396
1,985
Human infant faeces
CP002794, CP002795, and CP002796
B. longum
longum
longum
NCC2705
Complete
1
1
2,260
1,728
Human infant faeces
AE014295 and AF540971
B. longum
longum
longum
DJO10A
Complete
1
2
2,390
2,000
Human adult faeces
CP000605, AF538868, and AF538869
B. longumssp. longum
longum
longum
F8
Complete
1
2,385
1,681
Human feces
FP929034
B. longumssp. longum
longum
longum
ATCC 55813
Unfinished
140
2,373
2,109
Human infant faeces
ACHI00000000
B. longumssp. longum
longum
longum
CCUG 52486
Unfinished
55
2,453
2,240
Human elderly faeces
ABQQ00000000
B. longumssp. longum
longum
longum
1-6B
Unfinished
171
2,686
2,425
Human children feces
AJTF00000000
B. longumssp. longum
longum
longum
2-2B
Unfinished
141
2,625
2,412
Human children feces
AJTJ00000000
B. longumssp. longum
longum
longum
35B
Unfinished
131
2,514
2,260
Human infant faeces
AJTI00000000
B. longumssp. longum
longum
longum
44B
Unfinished
62
2,559
2,262
Human infant faeces
AJTM00000000
B. breve
breve
breve
ACS-071-V-Sch8b
Complete
1
2,327
1,826
Human vagina
CP002743
B. breve
breve
breve
UCC2003
Complete
1
2,423
1,852
Human infant faeces
CP000303
B. breve
breve
breve
DSM 20213
Unfinished
117
2,298
2,263
Human infant faeces
ACCG00000000
B. breve
breve
breve
CECT 7623
Unfinished
34
2,314
1,725
Human milk
AFVV00000000
B. breve
breve
breve
MCC0121
Unfinished
27
2,436
2,079
Human infant faeces
AVQA00000000
B. breve
breve
breve
MCC0305
Unfinished
22
2,287
1,927
Human adult faeces
AWFR00000000
B. breve
breve
breve
MCC0476
Unfinished
15
2,234
1,851
Human adult faeces
AVQB00000000
B. breve
breve
breve
MCC1094
Unfinished
23
2,327
1,944
Human infant faeces
AWFS00000000
B. breve
breve
breve
MCC1114
Unfinished
33
2,487
2,136
Human infant faeces
AVQC00000000
B. breve
breve
breve
MCC1128
Unfinished
25
2,480
2,143
Human infant faeces
AVQD00000000
B. breve
breve
breve
MCC1340
Unfinished
23
2,373
1,986
Human infant faeces
AWFT00000000
B. breve
breve
breve
MCC1454
Unfinished
14
2,457
2,111
Human infant faeces
AWFU00000000
B. breve
breve
breve
MCC1604
Unfinished
15
2,206
1,830
Human elderly faeces
AVQE00000000
B. breve
breve
breve
MCC1605
Unfinished
37
2,324
1,976
Human elderly faeces
AWFV00000000
B. animalisssp. animalis
animalis
animalis
ATCC 25527
Complete
1
1,933
1,538
Rat feces
CP002567
B. animalisssp. animalis
animalis
animalis
ATCC 27672
Unfinished
9
1,990
1,532
Rat feces
AWFQ00000000
B. animalisssp. animalis
animalis
animalis
MCC0483
Unfinished
27
2,176
1,750
Rat feces
AWFK00000000
B. animalisssp. animalis
animalis
animalis
MCC0499
Unfinished
11
2,134
1,721
Rat feces
AWFN00000000
B. animalisssp. animalis
animalis
animalis
MCC1489
Unfinished
14
1,910
1,474
Guinea pig feces
AWFO00000000
B. animalisssp. lactis
lactis
lactis
AD011
Complete
1
1,934
1,527
Human infant faeces
CP001213
B. animalisssp. animalis
lactis
lactis
ATCC 27536
Unfinished
18
1,912
1,561
Chicken feces
AWFL00000000
B. animalisssp. animalis
lactis
lactis
ATCC 27673
Unfinished
21
1,937
1,576
Sewage
AWFP00000000
B. animalisssp. animalis
lactis
lactis
ATCC 27674
Unfinished
18
1,912
1,561
Rabbit feces
AWFM00000000
B. animalisssp. lactis
lactis
lactis
B420
Complete
1
1
1,939
1,561
No information
CP003497
B. animalisssp. lactis
lactis
lactis
BB-12
Complete
1
1,942
1,642
Yoghurt
CP001853
B. animalisssp. lactis
lactis
lactis
Bi-07
Complete
1
1,939
1,597
No information
CP003498
B. animalisssp. lactis
lactis
lactis
Bl-04
Complete
1
1,939
1,567
Human infant faeces
CP001515
B. animalisssp. lactis
lactis
lactis
Bl12
Complete
1
1,938
1,518
Colonoscopic sample
CP004053
B. animalisssp. lactis
lactis
lactis
BLC1
Complete
1
1,944
1,518
No information
CP003039
B. animalisssp. lactis
lactis
lactis
BS01
Unfinished
7
1,932
1,572
No information
AHGW00000000
B. animalisssp. lactis
lactis
lactis
CNCM I-2494
Complete
1
1,943
1,660
No information
CP002915
B. animalisssp. lactis
lactis
lactis
DSM 10140
Complete
1
1,938
1,565
Yoghurt
CP001606
B. animalisssp. lactis
lactis
lactis
HN019
Unfinished
28
1,916
1,578
No information
ABOT00000000
B. animalisssp. lactis
lactis
lactis
V9
Complete
1
1,944
1,572
Human infant faeces
CP001892


## 2.2. Assimilation of Human Milk Oligosaccharides

Modified MRS was prepared by removing glucose from the original components and was supplemented with LNT (Dextra Laboratories Ltd., Reading, UK) at a final concentration of 2%. An aliquot of seed culture from each bifidobacterial strain was then inoculated into 200μL of the modified MRS, with a final concentration of 1%. Growth of each bifidobacterial strain was measured by absorbance at OD600 after cultivation under anaerobic conditions at 37°C for 24 h and 48 h. Experiments were performed in triplicate.


## 2.3. Genome Sequencing and Bioinformatics Analyses

Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The preparation of genomic libraries was performed with 1 ng of genomic DNA using the Nextera XT DNA Sample Preparation Kit (Illumina Inc., CA) according to the manufacturer’s instructions. After PCR amplification and cleanup, the fragment size distribution of the tagmented DNA was analysed using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA). The libraries were sequenced using a MiSeq Personal Sequencing System and the MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc.). Quality trimming andde novo assembly of the raw paired-end reads were performed using the CLC Genomics Workbench (v 6.0) software package (CLC bio, Aarhus, Denmark) with default settings, except for contig length (minimum contig length = 2,000 bp). Seventeen genomes that were sequencedde novo in this study were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2.0 and were manually checked as part of the process of genome submission to GenBank. Our sequencing efforts resulted in multiple contigs (Table 1). The datasets for the genome annotations for the other 32 strains published previously were retrieved from the FTP server of NCBI [20].


## 2.4. Pan-Genome Analysis

For all of the 49 bifidobacterial strains included in this study, a pan-genome calculation was performed using the PGAP [21]. The ORF content of each genome was organised into functional gene clusters using the gene family (GF) method. Under the GF method, the total protein sequences of each strain were mixed together, with each gene being marked as a strain identifier. BLASTALL searches were performed among the mixed protein sequences, and the filtered BLAST results were clustered using the Markov Cluster algorithm [22], which has been widely used in other studies on prokaryotic genomes and in programs designed to search for orthologues among multiple strains. For each gene pair in a given cluster, the global match region was no less than 50% of the longer gene protein sequence, and the identity was also no less than 50%. The minimum score value and E-value applied in BLAST were 50 and 1e-8. Pan-genome profile analysis, genetic variation analysis of functional genes, and function enrichment analysis of gene clusters were then performed. KEGG Orthology (KO) numbers were assigned by the KEGG Automatic Annotation Server (KAAS) using the bidirectional best-hit method [23].


## 2.5. Phylogenetic Trees

Pan-genome-based phylogenetic trees were generated according to a gene distance matrix, which was calculated based on genes that were absent or present in each strain [21], using UPGMA algorithms in PHYLIP. The sequences of 16S rRNA genes were aligned using the ClustalW alignment tool [24] with default parameters, and phylogenetic trees were constructed using UPGMA algorithms in MEGA6.06 [25]. Bootstrap values were calculated using 500 bootstrap replicates. The supertree was built using FigTree v1.4.0.


## 2.6. Nucleotide Sequence Accession Numbers

Thede novo sequence and annotation data reported herein have been deposited in GenBank under the accession numbers AVQA00000000-AVQE00000000 and AWFK00000000-AWFV00000000.


## 3. Results and Discussion

### 3.1. Phylogenetic Trees for Each Bifidobacterial Species

Based on the pan-genome profiles and 16S rRNA gene sequences, phylogenetic trees were constructed for each species, usingGardnerella vaginalis ATCC 14018 as an outgroup species. Each phylogenetic tree was divided into two clusters. However, the classification of subspecies of certain strains based on the genotypes of the strains was contradictory to that previously defined based on phenotypes (Table 1, Supplementary files 1–3 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/567809).B. animalis subsp.animalis ATCC 27536 and ATCC 27674 fell into the clade with the type strain ofB. animalis subsp.lactis, whereas B.longum subsp.longum JDM 301 andB. longum subsp.infantis 157F fell into the clades with the type strains ofB.longum subsp.infantis and B.longum subsp.longum, respectively. In this study, we adopted subspecies of these strains based on the phylogenetic trees for further analyses.


### 3.2. Core and Pan-Genome Structures

After clustering the functional genes for each species, respective totals of 5,471, 4,053, and 2,833 clusters and 966, 1,221, and 1,092 core clusters were obtained forB. longum,B. breve, andB. animalis (Supplementary file 4). These numbers are similar to those reported previously [11, 26, 27]. However, we identified a wider variety of genomes inB. longum compared with a previous report [11]. This result may suggest thatB. longum has an open pan-genome; that is,B. longum may exhibit a robust ability to import new genes, allowing it to adapt to each ecological niche over its long history of evolution. In the current study, to reveal differences among species, a total of 90,442 genes from the 49 strains were jointly clustered. These genes were divided into 8,818 homologous clusters, and there were 584 common clusters among all of the bifidobacterial strains (Figure 1). Additional analysis revealed that 404 of these clusters were commonly clustered withG. vaginalis ATCC 14018 as an outgroup species. Therefore, the number of specific gene clusters among the three bifidobacterial species was estimated to be less than 180. There were 166 common clusters between strains ofB. breve andB. longum, whereas there were nine common clusters between strains ofB. animalis andB. longum and four common clusters between strains ofB. animalis andB. breve (Figure 1).Figure 1
Venn diagram of the homologous clusters shared among the core genes. The surfaces are approximately proportional to the number of genes.


### 3.3. Metabolism of Host Glycans

Bifidobacteria have been reported to employ many unique metabolic pathways for sugar fermentation to enable them to utilise diverse carbohydrates in the intestine that are not utilised by their hosts [11–19]. Figure 2 shows the distributions of genes involved in the metabolism of host glycans, such as human milk oligosaccharides (HMOs), mucin, and N-glycans.Figure 2
Distribution of genes involved in the metabolism of host glycans, such as human milk oligosaccharides, mucin, and N-glycans. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


#### 3.3.1. HMOs

A 43 kb cluster in the genome ofB. longum subsp.infantis ATCC 15697 has been reported to be one of the most characteristic gene clusters for the utilisation of HMOs [28].Seven homologous genes encoding extracellular solute-binding proteins predicted to bind oligosaccharides (SBP family 1) were found in only two strains ofB. longumsubsp.infantis,whereas homologues of the major facilitator superfamily (Blon_2331, 2332) were found in all strains except forB. animalissubsp.lactis AD011. Two of four gene homologues encoding glycoside hydrolases (alpha-galactosidase and beta-N-acetylhexosaminidase) were found in strains ofB. breveas well asB. longum subsp.infantis.Fucosidase (Blon_2336) and sialidase (Blon_2348) homologues were found only inB. longumsubsp.infantis.However, certain strains ofB. breve possessed other gene clusters related to fucose and sialic acid incorporation (Figure 2).Regarding the core structure of HMOs, three of the four predominant HMOs (lacto-N-fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT)) exhibit the LNT structure. Therefore, the enzyme required for the utilisation of LNT is crucial in the utilisation of HMOs and the colonisation of the infant intestine. Certain strains belonging toB. bifidum, B. longum subsp.longum, B. longum subsp.infantis, andB. brevehave been reported to act as LNT consumers [29, 30]. The last two species incorporate intact LNT via an unidentified transporter and then hydrolyse it intracellularly using LNT β-1,3-galactosidase (Blon_2016) [31]. In our study, using type strains, we confirmed the ability ofB. longum subsp.longum, B. longum subsp.infantis, andB. breve, but notB. animalis subsp.animalis andB. animalis subsp.lactis, in the utilisation of LNT (Supplementary file 5). Unexpectedly, Blon_2016 homologues were also present inB. animalis strains. Based on phylogenetic analysis, Yoshida et al. [31] discovered that there is a close homologue of this gene (amino acid identity > 95%) that falls into a clade of strains of infant gut-related species (i.e.,B. breve,B. longumsubsp.infantis,andB. longumsubsp.longum), whereas the gene inB. animalis was observed to be distant from this clade. These results imply the existence of different substrate specificity for LNT β-1,3-galactosidase, although further investigations are necessary to examine this issue.B. bifidumand certain strains ofB. longum subsp.longumproduce a secretory enzyme, LNBase, that hydrolyses LNT into lacto-N-biose (LNB) and lactose [32, 33]. In the present study, homologous genes encoding LNBase (BLLJ_1506) and the chaperone for this enzyme (BLLJ_1505) were found in two strains ofB. longum subsp.longum. The LNBase ofB. longum subsp.longum has been reported to be able to hydrolyse the GlcNAcβ1-3Gal linkage in LNT, LNFP I, and sialyllacto-N-tetraose.These results imply the existence of different mechanisms for the utilisation of LNT decorated by fucose and sialic acid.B. bifidum might digest LNT after releasing its own fucosidase and sialidase, whereasB. longum subsp.longumlacks these enzymes but can directly degrade decorated LNT.Subsequently, the liberated LNB is imported into the cells via the galacto-N-biose (GNB)/LNB transporter for further degradation [34]. The disaccharide is then phosphorolysed by a GNB/LNB phosphorylase to produce Gal-1-P and GlcNAc and is further metabolised [34]. In the current study, a seven-gene operon involved in this pathway was observed in nearly all strains ofB. longumandB. breve and in three strains ofB. animalis subsp.animalis. This distribution is in complete accord with results regardingin vitro LNB utilisation [35]. A previous report revealing a strong ability to grow in human milk also supports these claims [36].


#### 3.3.2. Mucin and N-Glycans

Another substrate for this metabolic pathway is GNB, which is a core structure of gastrointestinal mucin. Mucins are extensively O-glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota. Studies have revealed that the main core structures of gastric/duodenal and intestinal mucins are core-1, 2-type and core-3-type O-glycans, respectively [37, 38]. A homologous gene encoding α-N-acetylgalactosaminidase, acting on core-3-type O-glycan (NagBb) [39], was observed in 2 strains ofB. longum subsp.infantis, 11 strains ofB. longum subsp.longum, and 13 strains ofB. breve in the present study. In contrast, a gene encoding endo-α-N-acetylgalactosaminidase, which is predicted to release GNB-containing glycans from core-1-type O-glycan mucin (BLD_1258) [40], was observed only inB. longum subsp.longum. In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [41, 42]. In our study, nearly all strains ofB. longum subsp.infantisandB. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine.Most strains ofB. longumandB. breve, but notB. animalisstrains, were found to possess genes involved in the pathway for the utilisation of N-acetylglucosamine (GlcNAc) and GalNAc, which are the major constituents of host glycans. Furthermore, gene homologues encoding endo-beta-N-acetylglucosaminidase (BLD_0197) [43], which releases complex N-glycans from human milk glycoproteins, and alpha-mannosidase (Blon_0868, 0869) were found in certain strains of these two species. These enzymes involved in the degradation of host glycans might play a role in the utilisation of intrinsic carbohydrate sources.Bifidobacteria are generally residents of the intestines of animals, including warm-blooded mammals and social insects, and several bifidobacterial species are typical inhabitants of the human gut (designated human-residential bifidobacteria or HRB). All strains ofB. longumandB. breve, which are typical HRB, possess certain genes related to the pathway for the utilisation of LNT/LNB, which are core structures of type I oligosaccharides that are specific to human breast milk (Figure 2). Among HRB strains in our study, nearly all possessed gene operons involved in the GNB/LNB pathway, whereas genes upstream of HMO utilisation, such as fucosidase, sialidase, and LNBase, were species/strain dependent. These results suggest that each HRB strain might evolve to assimilate HMOs, for which hundreds of types of structures have been reported [44]. In contrast,B. animalis is a non-HRB species; although certain strains ofB. animalis subsp.lactis have also been isolated from humans, they have been found in faecal samples but rarely in colon mucosal samples [27, 45]. In addition, previous reports have shown thatB. animalis is a strictly monophyletic group, and the evolutionary distance betweenB. animalis and species of HRB was shown to be relatively far based on 16S rRNA multigene alignments and comparative genomics [11, 13, 27, 46]. Our results indicate that there are fewer homologues involved in the degradation of host glycan byB. animalis(Figure 2). These results suggest that the observed differences in the gene distribution might be the result of the adaptation of these strains to their residential environments.


### 3.4. Carbohydrate-Active Enzymes for Plant-Derived Sugars

Regarding extrinsic carbohydrates, a variety of resistant fibres, which can be dietary compounds, are delivered to the colon, where bifidobacteria reside. Based on CAZy classification, all of the strains possessed a large number of homologous genes encoding GH 13 family members (Figure3), which has previously been reported as a characteristic feature of bifidobacterial genomes [47, 48]. These enzymes are typical enzymes for the degradation of alpha-glucopyranose units such as pullulan, starch, and amylopectin. In particular,B. longum subsp.longum was shown to be genetically well equipped for the fermentation of plant-derived sugars (Figure 3), which are assumed to be not introduced into the infant gut before weaning. A large number of GH 43 and 51 family members, which are enzymes responsible for the degradation of arabinose/xylose units such as arabinofuranoside and xylan, were found to be specific toB. longum subsp.longum, with strain specificity for several of these genes. Taken together with the distribution of genes for host glycan utilisation, such a wide range of genes for carbohydrate utilisation provides an advantage for colonisation of the human intestine byB. longum subsp.longum. This result might explain why onlyB. longum subsp.longum is a predominant species in both the infant and the adult intestines [49].Figure 3
Distribution of genes involved in the metabolism of plant-derived sugars predicted by the CAZy database. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


### 3.5. Membrane Transporters for Carbohydrate

KO assignment indicated several differences in the distribution of carbohydrate transporters at the subspecies level. The subspecies that possessed the greatest number of transporter homologues wasB. longum subsp.infantis (83.5±2.1), including taxon-specific transporters for GlcNAc, phosphonate, and urea, whereasB. animalis subsp.lactis possessed the lowest number of these homologues (33.7±1.3). All three species can assimilate fructose; however, only species ofB. longum exhibited a set of high-affinity fructose-specific ABC transporters (K02056, K02057, and K02058), which have been reported to be involved in efficiently converting fructose to acetate under certain gut conditions [5].B. longum subsp.longum lacked a homologous gene set for arabinogalactan transport. However, twelve strains ofB. longum subsp.longum possessed a set of homologous genes encoding extracellular enzymes for the degradation of arabinogalactan (BLLJ_1840) [50].B. brevealso exhibited certain unique ABC transporters for lactose/L-arabinose, ribose, and sn-glycerol 3-phosphate.We found distinctive differences between the three species in terms of the distribution of genes related to the phosphotransferase system (PTS), which is known to be a multicomponent system specific for sugar uptake that operates in global carbon regulation in many bacteria [51]. Strains ofB. longum andB. breve possessed three homologues, encoding phosphoenolpyruvate-protein kinase, phosphocarrier protein, and beta-glucoside-PTS, whereas none of theB. animalis strains exhibited these genes. Additionally, nearly all strains ofB. brevepossessed homologues of N-acetylglucosamine, glucose, fructose, and ascorbate, which are involved in the PTS (Figure 4).Figure 4
Distribution of genes encoding membrane transporters for carbohydrate. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


## 3.1. Phylogenetic Trees for Each Bifidobacterial Species

Based on the pan-genome profiles and 16S rRNA gene sequences, phylogenetic trees were constructed for each species, usingGardnerella vaginalis ATCC 14018 as an outgroup species. Each phylogenetic tree was divided into two clusters. However, the classification of subspecies of certain strains based on the genotypes of the strains was contradictory to that previously defined based on phenotypes (Table 1, Supplementary files 1–3 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/567809).B. animalis subsp.animalis ATCC 27536 and ATCC 27674 fell into the clade with the type strain ofB. animalis subsp.lactis, whereas B.longum subsp.longum JDM 301 andB. longum subsp.infantis 157F fell into the clades with the type strains ofB.longum subsp.infantis and B.longum subsp.longum, respectively. In this study, we adopted subspecies of these strains based on the phylogenetic trees for further analyses.


## 3.2. Core and Pan-Genome Structures

After clustering the functional genes for each species, respective totals of 5,471, 4,053, and 2,833 clusters and 966, 1,221, and 1,092 core clusters were obtained forB. longum,B. breve, andB. animalis (Supplementary file 4). These numbers are similar to those reported previously [11, 26, 27]. However, we identified a wider variety of genomes inB. longum compared with a previous report [11]. This result may suggest thatB. longum has an open pan-genome; that is,B. longum may exhibit a robust ability to import new genes, allowing it to adapt to each ecological niche over its long history of evolution. In the current study, to reveal differences among species, a total of 90,442 genes from the 49 strains were jointly clustered. These genes were divided into 8,818 homologous clusters, and there were 584 common clusters among all of the bifidobacterial strains (Figure 1). Additional analysis revealed that 404 of these clusters were commonly clustered withG. vaginalis ATCC 14018 as an outgroup species. Therefore, the number of specific gene clusters among the three bifidobacterial species was estimated to be less than 180. There were 166 common clusters between strains ofB. breve andB. longum, whereas there were nine common clusters between strains ofB. animalis andB. longum and four common clusters between strains ofB. animalis andB. breve (Figure 1).Figure 1
Venn diagram of the homologous clusters shared among the core genes. The surfaces are approximately proportional to the number of genes.


## 3.3. Metabolism of Host Glycans

Bifidobacteria have been reported to employ many unique metabolic pathways for sugar fermentation to enable them to utilise diverse carbohydrates in the intestine that are not utilised by their hosts [11–19]. Figure 2 shows the distributions of genes involved in the metabolism of host glycans, such as human milk oligosaccharides (HMOs), mucin, and N-glycans.Figure 2
Distribution of genes involved in the metabolism of host glycans, such as human milk oligosaccharides, mucin, and N-glycans. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


### 3.3.1. HMOs

A 43 kb cluster in the genome ofB. longum subsp.infantis ATCC 15697 has been reported to be one of the most characteristic gene clusters for the utilisation of HMOs [28].Seven homologous genes encoding extracellular solute-binding proteins predicted to bind oligosaccharides (SBP family 1) were found in only two strains ofB. longumsubsp.infantis,whereas homologues of the major facilitator superfamily (Blon_2331, 2332) were found in all strains except forB. animalissubsp.lactis AD011. Two of four gene homologues encoding glycoside hydrolases (alpha-galactosidase and beta-N-acetylhexosaminidase) were found in strains ofB. breveas well asB. longum subsp.infantis.Fucosidase (Blon_2336) and sialidase (Blon_2348) homologues were found only inB. longumsubsp.infantis.However, certain strains ofB. breve possessed other gene clusters related to fucose and sialic acid incorporation (Figure 2).Regarding the core structure of HMOs, three of the four predominant HMOs (lacto-N-fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT)) exhibit the LNT structure. Therefore, the enzyme required for the utilisation of LNT is crucial in the utilisation of HMOs and the colonisation of the infant intestine. Certain strains belonging toB. bifidum, B. longum subsp.longum, B. longum subsp.infantis, andB. brevehave been reported to act as LNT consumers [29, 30]. The last two species incorporate intact LNT via an unidentified transporter and then hydrolyse it intracellularly using LNT β-1,3-galactosidase (Blon_2016) [31]. In our study, using type strains, we confirmed the ability ofB. longum subsp.longum, B. longum subsp.infantis, andB. breve, but notB. animalis subsp.animalis andB. animalis subsp.lactis, in the utilisation of LNT (Supplementary file 5). Unexpectedly, Blon_2016 homologues were also present inB. animalis strains. Based on phylogenetic analysis, Yoshida et al. [31] discovered that there is a close homologue of this gene (amino acid identity > 95%) that falls into a clade of strains of infant gut-related species (i.e.,B. breve,B. longumsubsp.infantis,andB. longumsubsp.longum), whereas the gene inB. animalis was observed to be distant from this clade. These results imply the existence of different substrate specificity for LNT β-1,3-galactosidase, although further investigations are necessary to examine this issue.B. bifidumand certain strains ofB. longum subsp.longumproduce a secretory enzyme, LNBase, that hydrolyses LNT into lacto-N-biose (LNB) and lactose [32, 33]. In the present study, homologous genes encoding LNBase (BLLJ_1506) and the chaperone for this enzyme (BLLJ_1505) were found in two strains ofB. longum subsp.longum. The LNBase ofB. longum subsp.longum has been reported to be able to hydrolyse the GlcNAcβ1-3Gal linkage in LNT, LNFP I, and sialyllacto-N-tetraose.These results imply the existence of different mechanisms for the utilisation of LNT decorated by fucose and sialic acid.B. bifidum might digest LNT after releasing its own fucosidase and sialidase, whereasB. longum subsp.longumlacks these enzymes but can directly degrade decorated LNT.Subsequently, the liberated LNB is imported into the cells via the galacto-N-biose (GNB)/LNB transporter for further degradation [34]. The disaccharide is then phosphorolysed by a GNB/LNB phosphorylase to produce Gal-1-P and GlcNAc and is further metabolised [34]. In the current study, a seven-gene operon involved in this pathway was observed in nearly all strains ofB. longumandB. breve and in three strains ofB. animalis subsp.animalis. This distribution is in complete accord with results regardingin vitro LNB utilisation [35]. A previous report revealing a strong ability to grow in human milk also supports these claims [36].


### 3.3.2. Mucin and N-Glycans

Another substrate for this metabolic pathway is GNB, which is a core structure of gastrointestinal mucin. Mucins are extensively O-glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota. Studies have revealed that the main core structures of gastric/duodenal and intestinal mucins are core-1, 2-type and core-3-type O-glycans, respectively [37, 38]. A homologous gene encoding α-N-acetylgalactosaminidase, acting on core-3-type O-glycan (NagBb) [39], was observed in 2 strains ofB. longum subsp.infantis, 11 strains ofB. longum subsp.longum, and 13 strains ofB. breve in the present study. In contrast, a gene encoding endo-α-N-acetylgalactosaminidase, which is predicted to release GNB-containing glycans from core-1-type O-glycan mucin (BLD_1258) [40], was observed only inB. longum subsp.longum. In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [41, 42]. In our study, nearly all strains ofB. longum subsp.infantisandB. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine.Most strains ofB. longumandB. breve, but notB. animalisstrains, were found to possess genes involved in the pathway for the utilisation of N-acetylglucosamine (GlcNAc) and GalNAc, which are the major constituents of host glycans. Furthermore, gene homologues encoding endo-beta-N-acetylglucosaminidase (BLD_0197) [43], which releases complex N-glycans from human milk glycoproteins, and alpha-mannosidase (Blon_0868, 0869) were found in certain strains of these two species. These enzymes involved in the degradation of host glycans might play a role in the utilisation of intrinsic carbohydrate sources.Bifidobacteria are generally residents of the intestines of animals, including warm-blooded mammals and social insects, and several bifidobacterial species are typical inhabitants of the human gut (designated human-residential bifidobacteria or HRB). All strains ofB. longumandB. breve, which are typical HRB, possess certain genes related to the pathway for the utilisation of LNT/LNB, which are core structures of type I oligosaccharides that are specific to human breast milk (Figure 2). Among HRB strains in our study, nearly all possessed gene operons involved in the GNB/LNB pathway, whereas genes upstream of HMO utilisation, such as fucosidase, sialidase, and LNBase, were species/strain dependent. These results suggest that each HRB strain might evolve to assimilate HMOs, for which hundreds of types of structures have been reported [44]. In contrast,B. animalis is a non-HRB species; although certain strains ofB. animalis subsp.lactis have also been isolated from humans, they have been found in faecal samples but rarely in colon mucosal samples [27, 45]. In addition, previous reports have shown thatB. animalis is a strictly monophyletic group, and the evolutionary distance betweenB. animalis and species of HRB was shown to be relatively far based on 16S rRNA multigene alignments and comparative genomics [11, 13, 27, 46]. Our results indicate that there are fewer homologues involved in the degradation of host glycan byB. animalis(Figure 2). These results suggest that the observed differences in the gene distribution might be the result of the adaptation of these strains to their residential environments.


## 3.3.1. HMOs

A 43 kb cluster in the genome ofB. longum subsp.infantis ATCC 15697 has been reported to be one of the most characteristic gene clusters for the utilisation of HMOs [28].Seven homologous genes encoding extracellular solute-binding proteins predicted to bind oligosaccharides (SBP family 1) were found in only two strains ofB. longumsubsp.infantis,whereas homologues of the major facilitator superfamily (Blon_2331, 2332) were found in all strains except forB. animalissubsp.lactis AD011. Two of four gene homologues encoding glycoside hydrolases (alpha-galactosidase and beta-N-acetylhexosaminidase) were found in strains ofB. breveas well asB. longum subsp.infantis.Fucosidase (Blon_2336) and sialidase (Blon_2348) homologues were found only inB. longumsubsp.infantis.However, certain strains ofB. breve possessed other gene clusters related to fucose and sialic acid incorporation (Figure 2).Regarding the core structure of HMOs, three of the four predominant HMOs (lacto-N-fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT)) exhibit the LNT structure. Therefore, the enzyme required for the utilisation of LNT is crucial in the utilisation of HMOs and the colonisation of the infant intestine. Certain strains belonging toB. bifidum, B. longum subsp.longum, B. longum subsp.infantis, andB. brevehave been reported to act as LNT consumers [29, 30]. The last two species incorporate intact LNT via an unidentified transporter and then hydrolyse it intracellularly using LNT β-1,3-galactosidase (Blon_2016) [31]. In our study, using type strains, we confirmed the ability ofB. longum subsp.longum, B. longum subsp.infantis, andB. breve, but notB. animalis subsp.animalis andB. animalis subsp.lactis, in the utilisation of LNT (Supplementary file 5). Unexpectedly, Blon_2016 homologues were also present inB. animalis strains. Based on phylogenetic analysis, Yoshida et al. [31] discovered that there is a close homologue of this gene (amino acid identity > 95%) that falls into a clade of strains of infant gut-related species (i.e.,B. breve,B. longumsubsp.infantis,andB. longumsubsp.longum), whereas the gene inB. animalis was observed to be distant from this clade. These results imply the existence of different substrate specificity for LNT β-1,3-galactosidase, although further investigations are necessary to examine this issue.B. bifidumand certain strains ofB. longum subsp.longumproduce a secretory enzyme, LNBase, that hydrolyses LNT into lacto-N-biose (LNB) and lactose [32, 33]. In the present study, homologous genes encoding LNBase (BLLJ_1506) and the chaperone for this enzyme (BLLJ_1505) were found in two strains ofB. longum subsp.longum. The LNBase ofB. longum subsp.longum has been reported to be able to hydrolyse the GlcNAcβ1-3Gal linkage in LNT, LNFP I, and sialyllacto-N-tetraose.These results imply the existence of different mechanisms for the utilisation of LNT decorated by fucose and sialic acid.B. bifidum might digest LNT after releasing its own fucosidase and sialidase, whereasB. longum subsp.longumlacks these enzymes but can directly degrade decorated LNT.Subsequently, the liberated LNB is imported into the cells via the galacto-N-biose (GNB)/LNB transporter for further degradation [34]. The disaccharide is then phosphorolysed by a GNB/LNB phosphorylase to produce Gal-1-P and GlcNAc and is further metabolised [34]. In the current study, a seven-gene operon involved in this pathway was observed in nearly all strains ofB. longumandB. breve and in three strains ofB. animalis subsp.animalis. This distribution is in complete accord with results regardingin vitro LNB utilisation [35]. A previous report revealing a strong ability to grow in human milk also supports these claims [36].


## 3.3.2. Mucin and N-Glycans

Another substrate for this metabolic pathway is GNB, which is a core structure of gastrointestinal mucin. Mucins are extensively O-glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota. Studies have revealed that the main core structures of gastric/duodenal and intestinal mucins are core-1, 2-type and core-3-type O-glycans, respectively [37, 38]. A homologous gene encoding α-N-acetylgalactosaminidase, acting on core-3-type O-glycan (NagBb) [39], was observed in 2 strains ofB. longum subsp.infantis, 11 strains ofB. longum subsp.longum, and 13 strains ofB. breve in the present study. In contrast, a gene encoding endo-α-N-acetylgalactosaminidase, which is predicted to release GNB-containing glycans from core-1-type O-glycan mucin (BLD_1258) [40], was observed only inB. longum subsp.longum. In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [41, 42]. In our study, nearly all strains ofB. longum subsp.infantisandB. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine.Most strains ofB. longumandB. breve, but notB. animalisstrains, were found to possess genes involved in the pathway for the utilisation of N-acetylglucosamine (GlcNAc) and GalNAc, which are the major constituents of host glycans. Furthermore, gene homologues encoding endo-beta-N-acetylglucosaminidase (BLD_0197) [43], which releases complex N-glycans from human milk glycoproteins, and alpha-mannosidase (Blon_0868, 0869) were found in certain strains of these two species. These enzymes involved in the degradation of host glycans might play a role in the utilisation of intrinsic carbohydrate sources.Bifidobacteria are generally residents of the intestines of animals, including warm-blooded mammals and social insects, and several bifidobacterial species are typical inhabitants of the human gut (designated human-residential bifidobacteria or HRB). All strains ofB. longumandB. breve, which are typical HRB, possess certain genes related to the pathway for the utilisation of LNT/LNB, which are core structures of type I oligosaccharides that are specific to human breast milk (Figure 2). Among HRB strains in our study, nearly all possessed gene operons involved in the GNB/LNB pathway, whereas genes upstream of HMO utilisation, such as fucosidase, sialidase, and LNBase, were species/strain dependent. These results suggest that each HRB strain might evolve to assimilate HMOs, for which hundreds of types of structures have been reported [44]. In contrast,B. animalis is a non-HRB species; although certain strains ofB. animalis subsp.lactis have also been isolated from humans, they have been found in faecal samples but rarely in colon mucosal samples [27, 45]. In addition, previous reports have shown thatB. animalis is a strictly monophyletic group, and the evolutionary distance betweenB. animalis and species of HRB was shown to be relatively far based on 16S rRNA multigene alignments and comparative genomics [11, 13, 27, 46]. Our results indicate that there are fewer homologues involved in the degradation of host glycan byB. animalis(Figure 2). These results suggest that the observed differences in the gene distribution might be the result of the adaptation of these strains to their residential environments.


## 3.4. Carbohydrate-Active Enzymes for Plant-Derived Sugars

Regarding extrinsic carbohydrates, a variety of resistant fibres, which can be dietary compounds, are delivered to the colon, where bifidobacteria reside. Based on CAZy classification, all of the strains possessed a large number of homologous genes encoding GH 13 family members (Figure3), which has previously been reported as a characteristic feature of bifidobacterial genomes [47, 48]. These enzymes are typical enzymes for the degradation of alpha-glucopyranose units such as pullulan, starch, and amylopectin. In particular,B. longum subsp.longum was shown to be genetically well equipped for the fermentation of plant-derived sugars (Figure 3), which are assumed to be not introduced into the infant gut before weaning. A large number of GH 43 and 51 family members, which are enzymes responsible for the degradation of arabinose/xylose units such as arabinofuranoside and xylan, were found to be specific toB. longum subsp.longum, with strain specificity for several of these genes. Taken together with the distribution of genes for host glycan utilisation, such a wide range of genes for carbohydrate utilisation provides an advantage for colonisation of the human intestine byB. longum subsp.longum. This result might explain why onlyB. longum subsp.longum is a predominant species in both the infant and the adult intestines [49].Figure 3
Distribution of genes involved in the metabolism of plant-derived sugars predicted by the CAZy database. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


## 3.5. Membrane Transporters for Carbohydrate

KO assignment indicated several differences in the distribution of carbohydrate transporters at the subspecies level. The subspecies that possessed the greatest number of transporter homologues wasB. longum subsp.infantis (83.5±2.1), including taxon-specific transporters for GlcNAc, phosphonate, and urea, whereasB. animalis subsp.lactis possessed the lowest number of these homologues (33.7±1.3). All three species can assimilate fructose; however, only species ofB. longum exhibited a set of high-affinity fructose-specific ABC transporters (K02056, K02057, and K02058), which have been reported to be involved in efficiently converting fructose to acetate under certain gut conditions [5].B. longum subsp.longum lacked a homologous gene set for arabinogalactan transport. However, twelve strains ofB. longum subsp.longum possessed a set of homologous genes encoding extracellular enzymes for the degradation of arabinogalactan (BLLJ_1840) [50].B. brevealso exhibited certain unique ABC transporters for lactose/L-arabinose, ribose, and sn-glycerol 3-phosphate.We found distinctive differences between the three species in terms of the distribution of genes related to the phosphotransferase system (PTS), which is known to be a multicomponent system specific for sugar uptake that operates in global carbon regulation in many bacteria [51]. Strains ofB. longum andB. breve possessed three homologues, encoding phosphoenolpyruvate-protein kinase, phosphocarrier protein, and beta-glucoside-PTS, whereas none of theB. animalis strains exhibited these genes. Additionally, nearly all strains ofB. brevepossessed homologues of N-acetylglucosamine, glucose, fructose, and ascorbate, which are involved in the PTS (Figure 4).Figure 4
Distribution of genes encoding membrane transporters for carbohydrate. The abbreviations used for species/subspecies are as follows: INF,B. longumsubsp.infantis; LON,B. longum subsp.longum; BRE,B. breve; ANI,B. animalissubsp.animalis; LAC,B. animalissubsp.lactis.


## 4. Conclusions

Certain strains belonging to the three bifidobacterial species targeted in this study have been reported to exert numerous beneficial effects on human health as probiotics. Probiotic functionality is generally thought to be strain dependent. In fact, our analysis confirmed that certain genes, such as those encoding membrane transporters and enzymes for host glycan utilisation, are strain dependent. However, our data also demonstrated that there are common characteristics in each species that may be important in light of the species’ health-promoting effects on their hosts. Additionally, differences in characteristics might be a result of adaptation to the nutrient environments of each species (such as HRB versus non-HRB). Our results support previous observations based on the investigation of certain type strains of bifidobacterial species and enable the qualification of several of these characteristics as species common or strain specific [33, 39, 50, 52]. Taken together with other characteristics, such as vitamin metabolism [4, 53], colonisation factors [54], and extracellular components [55, 56], we believe that these findings will help to predict the features of probiotic strains. However, further study is needed to evaluate other non-HRB and HRB bifidobacterial species to attain a better understanding of the characteristics of these bacteria as well as the mechanisms underlying their residence in the host intestine and their potential functions as probiotics.



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