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. 2019 Apr 4;85(8):e03065-18. doi: 10.1128/AEM.03065-18

Unveiling Genomic Diversity among Members of the Species Bifidobacterium pseudolongum, a Widely Distributed Gut Commensal of the Animal Kingdom

Gabriele Andrea Lugli a,#, Sabrina Duranti a,#, Korin Albert b,c, Leonardo Mancabelli a, Stefania Napoli a, Alice Viappiani d, Rosaria Anzalone a, Giulia Longhi d, Christian Milani a, Francesca Turroni a,e, Giulia Alessandri f, David A Sela b,c,g, Douwe van Sinderen h, Marco Ventura a,e,✉
Editor: Andrew J McBaini
PMCID: PMC6450028  PMID: 30737347

Currently, little information exists on the genetics of the B. pseudolongum taxon due to the limited number of sequenced genomes belonging to this species. In order to survey genome variability within this species and explore how members of this taxon evolved as commensals of the animal gut, we isolated and decoded the genomes of 51 newly isolated strains. Comparative genomics coupled with growth profiles on different carbohydrates has further provided insights concerning the genotype and phenotype of members of the B. pseudolongum taxon.

KEYWORDS: Bifidobacterium, animal commensals, bifidobacteria, genomics, next-generation sequencing

ABSTRACT

Bifidobacteria are commensals of the animal gut and are commonly found in mammals, birds, and social insects. Specifically, strains of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium pseudolongum are widely distributed in the mammalian gut. In this context, we investigated the genetic variability and metabolic abilities of the B. pseudolongum taxon, whose genomic characterization has so far not received much attention. Phylogenomic analysis of the genome sequences of 60 B. pseudolongum strains revealed that B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum may actually represent two distinct bifidobacterial species. Furthermore, our analysis highlighted metabolic differences between members of these two subspecies. Moreover, comparative analyses of genetic strategies to prevent invasion of foreign DNA revealed that the B. pseudolongum subsp. globosum group exhibits greater genome plasticity. In fact, the obtained findings indicate that B. pseudolongum subsp. globosum is more adaptable to different ecological niches such as the mammalian and avian gut than is B. pseudolongum subsp. pseudolongum.

IMPORTANCE Currently, little information exists on the genetics of the B. pseudolongum taxon due to the limited number of sequenced genomes belonging to this species. In order to survey genome variability within this species and explore how members of this taxon evolved as commensals of the animal gut, we isolated and decoded the genomes of 51 newly isolated strains. Comparative genomics coupled with growth profiles on different carbohydrates has further provided insights concerning the genotype and phenotype of members of the B. pseudolongum taxon.

INTRODUCTION

The animal and bacterial kingdoms have coevolved and coadapted to establish interspecies relationships for hundreds of millions of years (1). The Human Microbiome Project and many other recent studies have achieved astounding progress in deciphering details on the human gut microbiota composition as well as its role in human health (2, 3). Members of the genus Bifidobacterium are prevalent and sometimes very abundant among the hundreds of bacterial species that inhabit the gut of humans and other mammals (2). Bifidobacteria are Gram-positive, non-spore-forming, and nonmotile bacteria that belong to the phylum Actinobacteria, and they represent one of the dominant microbial colonizers of the human and animal gut (4). Currently, 72 distinct bifidobacterial (sub)species are recognized, mostly isolated from the gastrointestinal tract of various animals, the human intestine and oral cavity, and insect hindgut (5–12). Previous genome analyses clearly indicate that bifidobacteria have developed a diverse number of genetic strategies to adapt to their specific ecological niches (5–12).

Recently, Milani et al. described bifidobacterial populations present among a wide range of 291 adult animals, thereby unveiling their widespread distribution across the mammalian kingdom (13). In this context, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium pseudolongum were shown to be the predominant bifidobacterial species present in the mammalian gut (13). To date, comprehensive comparative genomic analyses of these bifidobacterial taxa have been performed (14–16), with the notable exception of the B. pseudolongum species, for which currently little information exists with respect to its genomic characterization.

The B. pseudolongum species consists of two subspecies, B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum, of which B. pseudolongum subsp. pseudolongum was identified and classified in 1969 from swine feces (17). In the same year, Scardovi et al. isolated from the bovine rumen a strain they named to be Bifidobacterium globosum, which was further classified in 1992 as a subspecies of the B. pseudolongum species, i.e., B. pseudolongum subsp. globosum (18, 19). More recent studies have assessed the level of genome relatedness among the two subspecies of this taxon, casting doubt on the correct taxonomic classification of type strains previously assigned to these species (5, 20). Thus, in order to investigate the genetic variability and the metabolic capabilities of this taxon, 51 newly isolated B. pseudolongum strains from feces of various animals were subjected to de novo sequencing. The obtained genomic data sets were further supplemented with publicly available B. pseudolongum chromosomal sequences and were then subjected to in-depth comparative genomic analyses. Our findings revealed genotype and phenotype differences between the two subspecies of this taxon, highlighting two evolutionary routes that may be responsible for their differential host colonization preference.

RESULTS AND DISCUSSION

Isolation and genomic characterization of the B. pseudolongum taxon.

Recently, internal transcribed spacer (ITS) bifidobacterial profiling analysis was performed on fecal samples of various animals, revealing that B. pseudolongum is one of the most prevalent species (13). However, the genomic characterization of B. pseudolongum taxon is rather limited, and comparative genome analyses have not been reported that would characterize the genetic diversity and genomic features of members of this species. Thus, through a culture-dependent approach, we isolated 51 different B. pseudolongum strains from fecal samples of animals (Table 1). As shown in Table 1, we isolated B. pseudolongum strains from 22 mammalian species, as well as from birds, such as chicken (Gallus gallus domesticus), duck (Anas platyrhynchos domesticus), pigeon (Columba livia domestica), and quail (Coturnix coturnix). Exploring the bifidobacterial biodiversity among the mammalian gut microbiota, we were able to isolate strains from the following: (i) domesticated animals, e.g., alpaca (Vicugna pacos), camel (Camelus dromedarius), cow (Bos taurus), donkey (Equus africanus asinus), goat (Capra aegagrus hircus), horse (Equus ferus caballus), pig (Sus scrofa), and sheep (Ovis aries); and (ii) wild animals kept in captivity, such as bear (Ursus arctos), capybara (Hydrochoerus hydrochaeris), mara (Dolichotis patagonum), fox (Vulpes vulpes), hare (Lepus europaeus), hippopotamus (Hippopotamus amphibius), kangaroo (Macropus rufus), mouse (Mus musculus), porcupine (Erethizon dorsatum), mouflon (Ovis musimon), roe deer (Capreolus capreolus), and tapir (Tapirus terrestris). Furthermore, several canine breeds were investigated, allowing the isolation of eight B. pseudolongum strains, while a single strain was collected from a snake (Boa constrictor).

TABLE 1.

General genome features of B. pseudolongum strains

Strain Origin Cluster No. of bases Coverage (fold) No. of contigs No. of ORFs No. of rRNAs No. of tRNAs No. of TUGs HGT % CRISPR system RM system type Prophages Accession no.
Newly sequenced genomes
    22506a Goat glob-A 1,913,074 177 27 1,569 3 52 37 9.8 I-E 2 None RYUB00000000
    22511a Goat glob-A 2,018,875 233 35 1,645 3 52 62 11.9 I-E 3 None RYUA00000000
    102015a Cow glob-A 1,918,182 497 29 1,605 1 45 51 11 I-U 1 None RYTZ00000000
    102017a Cow glob-A 1,884,108 914 21 1,558 1 46 33 8.5 II 3 None RYTY00000000
    112206a Goat glob-A 1,947,489 851 29 1,613 1 47 54 10.8 None 5 None RYTX00000000
    1511Bb Chicken glob-B 1,971,056 152 11 1,604 3 51 37 11.2 I-E 1 None RYVN00000000
    1513Bb Piglet pseudo 1,855,765 124 6 1,532 3 52 34 8.3 I-C 3 None RYVM00000000
    1546Bb Chicken glob-B 1,955,522 68 15 1,635 2 53 13 10.8 I-E 1 1 RYVL00000000
    1550Bb Pigeon glob-B 2,000,909 204 5 1,659 4 53 32 11.6 II None 1 RYVK00000000
    1565Bb Donkey glob-B 1,956,592 266 15 1,638 2 53 12 11.2 I-E 1 1 RYVJ00000000
    1577Bb Horse glob-B 1,950,120 219 13 1,590 3 52 14 9.2 None 1 None SBKZ00000000
    1578Bb Duck glob-B 1,950,074 155 13 1,590 3 52 12 9.4 None 1 None RYVI00000000
    1604Bb Piglet pseudo 1,882,038 123 6 1,541 2 52 23 8.3 I-E 1 None RYVH00000000
    1612Bb Pig pseudo 1,891,497 155 5 1,583 3 53 38 7.8 None 1 1 RYVG00000000
    1616Bb Camel glob-A 2,031,794 246 39 1,676 2 52 49 10.1 I-E None 1 RYVF00000000
    1629Bb Tapir pseudo 1,852,430 156 6 1,542 1 52 30 9.5 None 5 None RYVE00000000
    1655Bb Alpaca glob-A 2,032,959 234 31 1,680 3 52 16 11 None 1 None SBKY00000000
    1678Bb Hippopotamus glob-A 2,037,960 84 34 1,680 3 52 21 11.1 None 2 None SBKX00000000
    1770Bb Fox glob-B 2,114,575 118 21 1,768 4 65 59 12.3 I-E 1 None RYVD00000000
    1780Bb Goat glob-A 1,981,485 177 27 1,618 2 52 39 11 None 4 None RYVC00000000
    1791Bb Roe deer glob-A 2,130,549 148 34 1,773 2 58 82 12.4 I-E 5 None RYVB00000000
    1805Bb Hare glob-B 2,102,705 113 12 1,729 3 52 58 12.6 I-U None 2 RYVA00000000
    2000Bb Dog glob-B 2,112,310 177 34 1,799 2 53 80 11.7 None 2 1 RYUZ00000000
    2001Bb Dog glob-B 2,095,316 137 21 1,736 3 52 62 11.8 II 2 1 RYUY00000000
    2002Bb Dog glob-B 2,057,959 271 20 1,722 2 54 43 9.7 I-C 2 1 RYUX00000000
    2003Bb Dog glob-B 2,148,604 145 37 1,816 3 59 77 12.7 I-C 1 2 RYUW00000000
    2004Bb Cow milk glob-A 1,910,716 305 17 1,571 2 53 32 9.4 None 4 None RYUV00000000
    2009Bb Dog glob-B 2,107,529 141 42 1,738 4 58 62 12.5 I-C 3 None RYUU00000000
    2012Bb Dog glob-B 2,070,775 215 35 1,753 3 53 82 13.2 None 3 2 SBKW00000000
    2017Bb Quail glob-B 2,189,937 178 12 1,891 3 60 20 14.7 II 2 2 RYUT00000000
    2019Bb Sheep glob-A 1,943,135 179 30 1,602 1 52 46 10.5 I-E 3 None RYUS00000000
    2023Bb Bear glob-B 1,983,829 214 19 1,636 2 53 23 10.6 None None 1 SBKV00000000
    2029Bb Hippopotamus glob-B 2,132,640 232 13 1,710 2 52 13 12.5 I-E 3 1 RYUR00000000
    2032Bb Capybara glob-B 2,132,192 250 14 1,709 2 52 12 12.4 I-E 3 1 RYUQ00000000
    2048Bb Pigeon glob-B 2,030,037 272 11 1,720 2 55 53 13.7 None 1 2 RYUP00000000
    2049Bb Quail glob-B 2,189,952 177 13 1,893 3 60 20 14.9 II 1 2 RYUO00000000
    2054Bb Dog pseudo 1,974,289 161 19 1,611 2 52 54 9.7 None 3 None RYUN00000000
    2071Bb Dog glob-B 2,084,649 247 36 1,767 3 60 73 12.8 I-C None 3 RYUM00000000
    2072Bb Hippopotamus glob-B 2,083,912 174 21 1,749 3 53 62 12.1 None 1 3 SBKU00000000
    2083Bb Sheep glob-A 2,059,818 131 20 1,679 3 53 45 9.8 II None None RYUL00000000
    2086Bb Hippopotamus glob-B 1,996,821 293 20 1,633 2 52 14 11.9 I-U 2 None RYUK00000000
    2088Bb Hippopotamus glob-B 1,991,771 295 17 1,633 1 52 14 11.4 I-U 2 None RYUJ00000000
    2089Bb Mara glob-B 2,047,482 322 9 1,633 2 52 22 10.8 II None None RYUI00000000
    2093Bb Snake glob-B 2,132,964 248 23 1,736 2 53 96 13 II 1 1 RYUH00000000
    2098Bb Mouflon glob-B 1,998,347 215 13 1,633 2 52 12 11.9 I-U 2 None RYUG00000000
    2103Bb Mouse glob-B 2,080,594 246 33 1,722 2 52 80 12.5 None None 1 SBKT00000000
    2105Bb Mouse glob-B 1,964,892 249 21 1,608 2 52 31 10.1 II 2 None RYUF00000000
    2109Bb Kangaroo glob-B 2,039,410 208 6 1,659 2 53 34 11.8 I-E None 1 SBKS00000000
    2113Bb Hippopotamus glob-B 1,996,348 218 15 1,631 2 52 13 11.8 I-U 2 None RYUE00000000
    2114Bb Porcupine glob-B 2,047,198 273 19 1,646 2 52 22 11.7 II None None RYUD00000000
    2115Bb Porcupine glob-B 2,016,784 329 12 1,627 3 52 17 11.5 II None None RYUC00000000
Public genomes
    DSM20092 Cow glob-A 1,978,157 NAc 1 1,598 4 52 21 9.1 None 1 None CP017695
    LMG11569 Cow glob-A 1,935,255 NA 26 1,565 3 52 29 9.2 None 3 None JGZG00000000
    LMG11571 Pig pseudo 1,898,684 NA 11 1,494 3 52 61 8.8 I-E 1 None JGZH00000000
    PV8-2 Human infant glob-B 2,032,698 NA 1 1,685 4 52 47 11.8 I-U None 1 CP007457
    UMB-MBP-01 Mouse glob-B 2,008,102 NA 1 1,645 4 52 56 10.5 II 1 1 CP022544
    1370B Pig pseudo 1,902,036 NA 17 1,582 5 52 54 10.8 None 2 None PCHI00000000
    1520B Hamster glob-B 2,008,481 NA 19 1,630 4 52 59 10.6 None 1 None PCHH00000000
    1549B Brahma chicken glob-B 1,959,452 NA 19 1,647 2 53 16 11.4 I-E 1 1 PCHG00000000
    1595B Pig pseudo 1,936,418 NA 16 1,603 4 53 67 10.4 None 2 None PCHF00000000
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a

Genome sequences decoded using a NextSeq platform (Illumina, UK).

b

Genome sequences decoded using a MiSeq platform (Illumina, UK).

c

NA, not applicable.

Accordingly, the genomes of these 51 newly isolated B. pseudolongum strains were decoded by means of a next-generation sequencing (NGS) approach, and the obtained data were analyzed together with nine publicly available B. pseudolongum sp. genomes (Table 1). All B. pseudolongum genomes of newly isolated strains were sequenced to a coverage depth that ranged from 68-fold to 914-fold, which upon assembly resulted in five to 42 contigs (Table 1). We were able to predict the contig orientation and the order for each draft genome using the complete genome of the type strain B. pseudolongum DSM 20092 (NCBI accession no. CP017695) as a reference sequence. The individual genome length of each B. pseudolongum strain was retrieved by the assembly, resulting in genomes whose sizes were shown to range from 1,852,430 to 2,189,952 bp (Table 1). As outlined in Table 1, the number of predicted open reading frames (ORFs) in each genome ranged from 1,494 ORFs for B. pseudolongum subsp. pseudolongum LMG 11571 to 1,893 ORFs for B. pseudolongum subsp. globosum 2049B, displaying a higher average number of ORFs in genomes of isolates that belong to the B. pseudolongum subsp. globosum taxon (Table 1). Furthermore, 81% of the predicted B. pseudolongum ORFeome was functionally classified on the basis of the eggNOG database (21). A large proportion of classified genes was predicted to be involved in housekeeping functions, amino acid and carbohydrate metabolism, and associated transport activities (Fig. 1a), resembling the functional content in genomes of other members of the Bifidobacterium genus (6, 20, 22, 23).

FIG 1.

FIG 1

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B. pseudolongum pangenome and core genome. (a) Number of core genes (green), unique genes (blue), and dispensable genes (yellow) identified in the pangenome analysis (internal pie chart). COG classifications of the whole B. pseudolongum pangenome are highlighted in different colors (external pie chart). (b) Pangenome size based on sequential addition of the 60 B. pseudolongum genomes. (c) Core genome size based on sequential addition of the B. pseudolongum genomes.

Pangenome and core genome of the B. pseudolongum species.

All identified genes for each B. pseudolongum isolate were employed for a comparative genome analysis of this species. Thus, pangenome and core-genome analyses of this taxon were undertaken following a previously described method based on Clusters of Orthologous Groups (COGs) (24). This analysis resulted in the identification of 6,179 COGs, representing the pangenome of the B. pseudolongum species. Notably, 1,069 COGs of this pangenome were shared among the 60 B. pseudolongum genomes, thus representing the so-called core genome of this taxon (Fig. 1c). Furthermore, dispensable genes present in two or more (but not all) strains, and truly unique genes (TUGs) which are present in just one of the analyzed strains were also identified. TUGs of each B. pseudolongum strain, ranging from 12 for B. pseudolongum 1565B to 96 for B. pseudolongum 2093B, were detected, with an average of 41 TUGs per genome. When this number is compared to other previously analyzed bifidobacterial pangenomes, such as Bifidobacterium adolescentis and Bifidobacterium bifidum (14, 15), it appears that B. pseudolongum ORFeome contains a relatively small number of TUGs, indicating that we have sufficiently explored the variability within this taxon.

The pangenome size, when plotted versus the number of included bifidobacterial genomes, shows that the power trendline tends to reach a plateau, where genomic data from the last strain added to the analysis do not substantially expand the total gene pool (Fig. 1b). Therefore, according to these data, the resulting pangenome curve suggests a “closed” B. pseudolongum pangenome. This means that after addition of the 60th B. pseudolongum genome, any further genome additions will result in only minor increases in the pangenome. In the same fashion, a pangenome was previously disclosed for Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium longum species (16, 25, 26).

Phylogenomic analyses and evolutionary development of B. pseudolongum taxon.

The comparative genomic analysis also allows the assessment of phylogeny of the B. pseudolongum taxon, applying a previously described methodology (20, 22, 27). To perform such a phylogenomic analysis, the genomes of the 60 B. pseudolongum strains were used for this exercise, as well as the gene sequence of B. animalis subsp. animalis ATCC 25527, which served as an outgroup. Furthermore, paralogs were excluded from the 1,069 B. pseudolongum core genes identified and described above by means of PGAP analysis (see Materials and Methods), resulting in 1,038 genes of which the concatenated amino acid sequences were employed to build a supertree (Fig. 2). The resulting B. pseudolongum-based phylogenomic supertree clearly shows the presence of two major groups which were assumed to correspond to the subspecies of this taxon. Within the supertree, the type strain B. pseudolongum subsp. pseudolongum LMG 11571 shared the same phylogenetic branch as that of seven B. pseudolongum strains, while the type strain B. pseudolongum subsp. globosum LMG 11569 was positioned in the second branch together with the remaining 52 strains (Fig. 2). Thus, our isolation attempts from stool samples of animals indicates that strains belonging to the B. pseudolongum subsp. globosum group outnumber members of the B. pseudolongum subsp. pseudolongum group.

FIG 2.

FIG 2

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Phylogenomic tree of the B. pseudolongum taxon. (a) Proteomic tree based on the concatenation of 1,038 core genes identified in the pangenome analysis of the 60 B. pseudolongum strains. Phylogenetic groups are highlighted in different colors. The tree was constructed by the neighbor-joining method, and the genome sequence of B. animalis subsp. animalis ATCC 25527 was used as an outgroup. Bootstrap percentages above 50 are shown at node points, based on 1,000 replicates. (b) Relative abundance of B. pseudolongum within analyzed animals based on ITS bifidobacterial profiling. n.d., not detected.

Interestingly, strains of the smaller cluster, represented by members of the B. pseudolongum subsp. pseudolongum group, were all isolated from fecal samples of pigs, with the exception of the 1629B and 2054B strains that were retrieved from tapir and dog fecal samples, respectively (Fig. 2). Furthermore, none of the 52 strains belonging to the B. pseudolongum subsp. globosum group were of porcine origin, a finding that suggests that members of the B. pseudolongum subsp. pseudolongum taxon reside in a specific ecological niche. The B. pseudolongum subsp. globosum group also appeared to include a conserved subcluster composed of 15 strains (Fig. 2). Remarkably, and similar to the B. pseudolongum subsp. pseudolongum group, members of this subcluster were isolated from specific animals in which the other 37 strains of the B. pseudolongum subsp. globosum group had not been detected. In this context, the hosts of this subcluster were represented by several ruminant species, such as cow, goat, sheep, camel, and alpaca, perhaps highlighting a genomic adaptation of these B. pseudolongum strains to a diet rich in grass-based fiber content. In contrast, the remaining strains of the B. pseudolongum subsp. globosum group were isolated from a wide selection of mammals, both herbivorous and carnivorous, and including birds, such as chicken, pigeon, quail, and duck (Fig. 2). Thus, members of the B. pseudolongum species appear to be widespread among the animal kingdom, but it appears that different animals host a specific cluster of members of this taxon.

Using the average nucleotide identity (ANI) approach, between all collected B. pseudolongum pairs, we highlighted the genome synteny among members of this species, with associated ANI values ranging from 92.7% to 99.9% (see Table S1 in the supplemental material). Notably, two strains that display an ANI value of <95% may be considered to belong to two distinct species (28). In this regard, in our previous phylogenomic studies concerning the Bifidobacterium genus, we applied an ANI threshold of about 94% to discriminate between bifidobacterial species (6, 20, 27). Assessment of ANI values among members of a given B. pseudolongum subspecies showed that B. pseudolongum subsp. pseudolongum members exhibit higher values (greater than 98.4%) than those of the B. pseudolongum subsp. globosum group (greater than 94.8%) (Table S1). Thus, despite the absence of a subspecies-specific core genome within the species, phylogenomic analyses highlight extensive variability between genomes of members of the two groups. Altogether, our findings suggest that B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum represent distinct bifidobacterial species rather than two separate subspecies.

Genome plasticity of the B. pseudolongum species.

In order to identify genes that may have been acquired by horizontal gene transfer (HGT), the genomes of the 60 B. pseudolongum strains were screened using the software tool COLOMBO (see Materials and Methods), resulting in the identification of HGT genes that make up 7.8% of the total gene pool in the case of B. pseudolongum 1612B and up to 14.9% of that of B. pseudolongum 2049B (Table 1). The functional classification based on the eggNOG database revealed that just 45% of the predicted alien genes could be functionally assigned (Fig. S2). Furthermore, unlike the whole proteome of the B. pseudolongum taxon, 44% of these genes are of unknown function, followed by genes predicted to encode replication and repair systems and cell wall and membrane proteins (Fig. S2). Interestingly, members of the B. pseudolongum subsp. pseudolongum group exhibit a lower average percentage of predicted HGT events (9.2%), followed by members of the ruminant subcluster of B. pseudolongum subsp. globosum group (10.4%) and the remaining strains (11.8%). Thus, B. pseudolongum subsp. globosum strains that were isolated from a wider number of animals seem to possess an enhanced propensity to acquire alien genes. In a similar fashion, the length of the analyzed genomes and their corresponding number of dispensable genes reflect the same trend. In fact, the average length of the B. pseudolongum subsp. pseudolongum genomes is significantly lower than that of the B. pseudolongum subsp. globosum (P < 0.001), i.e., 1.89 and 2.03 Mb, respectively (Table 1), while the number of dispensable genes increases to 21% in members of the B. pseudolongum subsp. globosum group. Thus, a correlation seems to exist between genome size, number of dispensable genes, and ecological niches of the analyzed B. pseudolongum strains.

It has already been shown that prophage sequences represent a large part of the bifidobacterial genetic repertoire acquired by HGT (22). We therefore performed a prophage profiling of the pangenome of the B. pseudolongum by means of previous bifidophage sequences classified by Lugli et al. (29). This genomic screen revealed the presence of 35 complete prophage sequences harbored by 25 B. pseudolongum strains (Table 1). In this context, the retrieved prophage sequences were predicted to belong to the Siphoviridae family and were shown to include modules that putatively encode functions involved in lysogeny, DNA replication, DNA packaging, head and tail morphogenesis, and host lysis (30). Furthermore, 10 apparently incomplete prophage sequences, i.e., prophages that exhibit extensive genome degeneration, were identified in just as many strains. Overall, this analysis showed that complete prophages were retrieved from B. pseudolongum subsp. globosum strains only, with the exception of one prophage sequence contained in the B. pseudolongum subsp. pseudolongum 1612B genome (Table 1). Moreover, among members of the B. pseudolongum subsp. globosum taxon, 33 out of 34 prophage sequences were identified in strains belonging to the wider phylogenetic cluster (Fig. 2).

Defense mechanisms of the B. pseudolongum species.

In order to investigate the ability of members of the B. pseudolongum group to defend themselves against invasion by foreign DNA, we investigated the presence of active CRISPR-Cas systems, as previously reported for the type strains of the genus Bifidobacterium by Briner et al. (31). Among the 60 B. pseudolongum genomes analyzed, we observed a high percentage of CRISPR-Cas system occurrence (67%) (Tables 1 and S2). According to the cas gene content and CRISPR length, we identified 28 type I systems and 12 type II systems, while type III systems appear to be absent in our assessed strain collection (Fig. S2). In this context, the lower number of type II systems, compared to the type I systems, reflects the previously stated notion that type II systems are fairly rare in nature, occurring in just 5% of currently known bacteria (32). When scrutinizing the CRISPR sequences, 14 different repeats were identified, of which each sequence corresponds to a specific (sub)type system, e.g., I-E, I-C, and I-U. Interestingly, strains 2009B and 2093B possess two different CRISPR sequences, yet only one CRISPR-Cas system appears to be complete, being type I-C and type II, respectively (Table S2). Type I systems, including the I-E, I-C, and I-U subtypes, were the most commonly encountered CRISPR-Cas systems and the only ones identified in the B. pseudolongum subsp. pseudolongum cluster. In contrast, type II systems were identified only in members of the B. pseudolongum subsp. globosum cluster. Furthermore, the type II system distribution among the B. pseudolongum strains, based on the phylogenomic tree of this taxon, highlighted a more recent acquisition of this system with respect to type I systems (Fig. 2). Moreover, genome sequencing of the B. pseudolongum strains allowed the identification of prophages matching spacer sequences of CRISPR loci identified in the study. Strains possessing a CRISPR-Cas system displayed at least one match with prophage sequences with an identity value ranging from 97% to 100% (Table S3). Interestingly, both subspecies seem to have acquired immunity through these CRISPR-Cas systems against B. pseudolongum subsp. globosum-derived prophages retrieved from strains of the wide cluster (94% of the hits) (Table S3).

Other multiprotein complexes that prevent acquisition of foreign DNA are represented by restriction-modification (RM) systems (33). Screening the predicted B. pseudolongum proteome for RM systems revealed that type I RM systems represent the predominant gene cluster, being present in 35 strains, followed by type II RM systems identified on the genomes of 25 B. pseudolongum strains (Tables 1 and S4). Besides, type III and IV RM systems were identified at a lower frequency, i.e., in six and 16 strains, respectively (Fig. S2). Remarkably, members of the B. pseudolongum subsp. pseudolongum group encoded an average of 2.3 complete RM systems per genome, while B. pseudolongum subsp. globosum group encoded an average of only 1.5 systems. Thus, it appears that the genomes of B. pseudolongum species isolated from pigs were equipped with a wider genomic arsenal to prevent the invasion by foreign DNA sequences.

Taken together, these findings indicate a higher occurrence of prophage sequences in members of the B. pseudolongum group, which possess a lower number of genetic clusters encoding proteins aimed to defend themselves against foreign DNA invasion (Table 1). Thus, our analyses revealed a correlation between a lower number or RM/CRISPR systems and an increase of dispensable genes of the B. pseudolongum subsp. globosum taxon, similar to what was recently shown for the B. breve taxon (34). Accordingly, the higher genome plasticity of members of the B. pseudolongum subsp. globosum group, perhaps as a result of the lower abundance of RM/CRISPR systems, may explain an enhanced ability to adapt to different ecological niches such as mammals and birds.

B. pseudolongum carbohydrate-active enzymes and growth profiles on different carbohydrates.

To identify the carbohydrate-active enzyme repertoire of each analyzed B. pseudolongum genome, we investigated the presence of genes predicted to encode glycosyl hydrolases (GHs). This analysis identified members of 28 GH families, highlighting a predominance of genes encoding GHs belonging to the GH13, GH43, and GH36 families, predicted to be responsible for the breakdown of plant-derived polysaccharides, such as starch, and a wide range of carbohydrates, e.g., maltodextrin, melibiose, and raffinose (Fig. 3). Together with GH2, GH3, GH25, GH30, GH51, and GH77 families, which were also identified in the glycobiome of each B. pseudolongum strain, these GH families represent the core glycobiome of the B. pseudolongum taxon. The B. pseudolongum subsp. pseudolongum group was shown to encode a higher number of GH112 than did the B. pseudolongum subsp. globosum group, encoding lacto-N-biose phosphorylase. In contrast, the glycobiome of members of the B. pseudolongum subsp. globosum group unveiled a higher number of GH29, GH31, and GH42 families, encoding putative α-l-fucosidase, α-glucosidase, and β-galactosidase activity, respectively (Fig. 3). Furthermore, analysis of the glycobiomes of members of the B. pseudolongum subsp. globosum group isolated from ruminant species revealed that GH1 and GH94 members are rare compared to other strains belonging to B. pseudolongum subsp. globosum. Altogether, members of the B. pseudolongum subsp. pseudolongum group and the ruminant subcluster of the B. pseudolongum subsp. globosum taxon possessed a lower number of genes encoding GHs than did the remainder of the B. pseudolongum strains analyzed here. Accordingly, B. pseudolongum subsp. globosum strains that were not isolated from ruminants revealed an extensive variability in GH enzymes which would be consistent with their ability to colonize a wider number of different animal hosts (Fig. 3).

FIG 3.

FIG 3

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Predicted glycobiome of the B. pseudolongum species and relative growth performances. The top of the image represents the predicted glycobiome of the 60 B. pseudolongum strains, while the heat map at the bottom of the image depicts growth performances of B. pseudolongum strains on different sugars. N-acetyl-d-Galact, N-acetyl-d-galactosamine; N-acetyl-d-Glucos, N-acetyl-d-glucosamine.

In order to validate the above-described genomic-based analyses, we carried out growth experiments of B. pseudolongum species strains on 27 carbohydrates including host- and plant-derived glycans as the sole carbon source (Fig. 3). As displayed in Fig. 3, all B. pseudolongum subsp. pseudolongum strains were able to grow on several sugars, such as glucose, glycogen, lactose, maltodextrin, maltose, melibiose and raffinose. In contrast, fermentation capabilities of carbohydrates were shown to be quite varied for B. pseudolongum subsp. globosum. However, statistical analyses were performed to corroborate the observed growth differences between B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum strains on different sugars. As shown in Fig. 4, the comparison of metabolic capabilities between B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum strains showed a significant growth difference (P < 0.05) for 18 carbohydrates. Specifically, B. pseudolongum subsp. globosum strains displayed higher growth performances when the growth medium was supplemented with cellobiose, rhamnose, starch, trehalose, N-acetyl-d-galactosamine, or N-acetyl-d-glucosamine. On the other hand, B. pseudolongum subsp. pseudolongum strains were shown to grow significantly better in medium supplemented with glycogen, glucose, pullulan, maltose, ribose, lactose, sucrose, maltodextrin, melibiose, turanose, fructose, mannitol, or lacto-N-tetraose (Fig. 4). As expected, the higher growth performance of B. pseudolongum subsp. pseudolongum grown in the presence of these carbohydrates is consistent with the abundance of GH112 as unveiled by the in silico glycobiome analysis (Fig. 3). Interestingly, in contrast to B. pseudolongum subsp. pseudolongum strains, members of the B. pseudolongum subsp. globosum subcluster display limited growth on glucose and lactose. Strains of the B. pseudolongum subsp. globosum subcluster have been isolated from ruminants, which are likely to have a rather low abundance of simple sugars, such as glucose and lactose, in the large intestine since most of these carbohydrates are metabolized in the rumen (35). Thus, members of the B. pseudolongum subsp. globosum subcluster may have evolved genetic capabilities toward the metabolism of complex dietary carbohydrates that end up in the large intestine of ruminants without being hydrolyzed (35).

FIG 4.

FIG 4

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Statistical analysis based on the carbohydrate growth assays. Whisker plot based on optical density values of those sugars that results in a P value of <0.05 between subspecies (Student’s t test). The x axis represents the sole carbon source used for the growth experiments, while the y axis shows the optical density values obtained for B. pseudolongum subsp. pseudolongum strains (green) and B. pseudolongum subsp. globosum strains (blue). Dots reflect the distribution of a data set, while the boxes represent 50% of the data set, distributed between the 1st and 3rd quartiles. The median divides the boxes into the interquartile range, while the X represents the mean. The lines extending vertically outside the boxes show the outlier range.

Conclusions.

The current study provides insights regarding the genotype and phenotype of members of the B. pseudolongum taxon. The genome sequencing of 51 newly isolated strains from mammals and birds allowed us to perform a comparative genomic analysis unveiling the genetic makeup of members of the B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum subspecies. Phylogenomic analyses highlighted a phylogenetic cluster consisting of eight strains and representing members of the B. pseudolongum subsp. pseudolongum group that were exclusively isolated from the gut of pigs. Conversely, members of the B. pseudolongum subsp. globosum cluster were isolated from a plethora of animals, reflecting their relatively (compared to B. pseudolongum subsp. pseudolongum) high genome variability and providing support for a possible taxonomic reclassification of these subspecies into two separate species. Furthermore, in silico analyses revealed an apparently higher genome plasticity in members of the B. pseudolongum subsp. globosum group. B. pseudolongum subsp. pseudolongum strains, on the other hand, showed a higher number of defense systems to prevent foreign DNA invasion, perhaps also explaining their smaller genomes. Moreover, differences between subspecies were highlighted through the prediction of their glycobiome and growth profiles on different carbohydrates. Altogether, these results highlight that B. pseudolongum subsp. pseudolongum strains evolved as commensals of a specific ecological niche, trying to minimize the acquisition of alien DNA, while members of the B. pseudolongum subsp. globosum group evolved in an opposite direction in order to colonize the gastrointestinal tract of a wide range of animals.

MATERIALS AND METHODS

Bifidobacterial genome sequences.

We retrieved the complete genome sequences of three B. pseudolongum strains from the National Center for Biotechnology Information (NCBI) public database. In the same fashion, partial genome sequences of six B. pseudolongum strains were retrieved from the NCBI in order to include the type strain of both subspecies and assembled genomes that consisted of <20 contigs.

Recovery of bifidobacterial strains and growth conditions.

One gram of a given fecal sample was mixed with 9 ml of phosphate-buffered saline (PBS; pH 6.5). Serial dilutions and subsequent platings were performed using de Man-Rogosa-Sharpe (MRS) agar (Scharlau Chemie, Barcelona, Spain) supplemented with 0.05% (wt/col) l-cysteine hydrochloride and 50 μg/ml mupirocin (Delchimica, Italy). The agar plates were incubated in an anaerobic atmosphere (2.99% H2, 17.01% CO2, and 80% N2) in a chamber (Concept 400; Ruskin) at 37°C for 48 h. Approximately 3,500 colonies were selected and were subcultivated in MRS broth supplemented with 0.05% (wt/col) l-cysteine hydrochloride in anaerobic chamber at 37°C for 16 h. DNA was extracted using GenElute bacterial genomic DNA kits (Sigma-Aldrich) following the manufacturer’s guide and then subjected to a B. pseudolongum-specific species identification PCR using primers Blong1 (5′-TTCCAGTTGATCGCATGGTC-3′) and Blong2 (5′-GGGAAGCCGTATCTCTACGA-3′), which had been designed on the 16S rRNA gene sequence of this species. PCR products were detected with SYBR safe DNA gel stain after amplification according to the following protocol: one cycle of 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 50 s, and finally one cycle of 72°C for 5 min. The isolated strains employed in this study are listed in Table 1. Furthermore, in order to discriminate between subspecies, PCR analysis were performed using DNA extracted from fecal samples of animals, as previously described by Milani et al. (13). The specific primers for the B. pseudolongum subsp. pseudolongum taxon are Bpseudolo_F (5′-CAGGCGTTCCTGTGGTTC-3′) and Bpseudolo_R (5′-GCGATGATGGCGAATGAC-3′), while primers B.p.glob_F (5′-GCAAGTCTCCAATGTTGAGG-3′) and B.p.glob_R (5′-CTGTGCGGACGAGACGTAG-3′) were used for the B. pseudolongum subsp. globosum taxon. Amplicons were detected with SYBR safe DNA gel stain after amplification according to the following protocol: one cycle of 94°C for 5 min, followed by 30 cycles of 94°C for 20 s, 54°C or 58°C for 20 s, and 72°C for 30 s, and finally one cycle of 72°C for 5 min.

Chromosomal DNA extraction.

In order to perform chromosomal DNA extraction, B. pseudolongum strains were inoculated in de Man-Rogosa-Sharpe (MRS; Scharlau Chemie) medium supplemented with 0.05% (wt/vol) l-cysteine hydrochloride and incubated at 37°C in an anaerobic atmosphere (2.99% [vol/vol] H2, 17.01% [vol/vol] CO2, and 80% [vol/vol] N2) using an anaerobic chamber (Concept 400; Ruskin). Cells from 10 ml of an overnight culture were harvested by centrifugation at 6,000 rpm for 8 min, and the obtained cell pellet was used for DNA extraction using the GenElute bacterial genomic DNA kit (Sigma-Aldrich) following the manufacturer’s guidelines. Internal transcribed spacer (ITS) sequences were amplified from extracted DNA using primer pair Probio-bif_Uni/Probio-bif_Rev (36) and sequenced to avoid decoding of clonal strains.

Genome sequencing and assemblies.

The genome sequences of 46 B. pseudolongum strains were determined by GenProbio srl (Parma, Italy) using a MiSeq platform (Illumina, UK). A genome library was generated using the TruSeq Nano DNA kit following a specified protocol (part no. 15041110 rev. D). The generated library samples were then loaded into a 600-cycle flow cell version 3 (Illumina). The remaining five B. pseudolongum strains were sequenced on the Illumina NextSeq platform with NextSeq V2 reagents using a Nextera XT 150-bp paired-end library preparation kit (catalog no. FC-131-1096), as per the manufacturer’s instructions. Fastq files of paired-end reads obtained from each individual genome sequencing effort were used as input for the genome assembly through the MEGAnnotator pipeline (37). The SPAdes program (version 3.12.0) was used for de novo assembly of each bifidobacterial genome sequence with the pipeline option “–carefull” and a list of k-mer sizes of 21,33,55,77,99,127 (38). Contigs greater than 1,000 bp were then employed by MEGAnnotator for the prediction of protein-encoding open reading frames (ORFs) using Prodigal (39). Predicted ORFs were functionally annotated by means of RAPSearch2 (Reduced Alphabet based Protein similarity Search) (cutoff E value, 1 × 10−5; minimum alignment length, 20 amino acids) performed against the NCBI nr database (40) coupled with hidden Markov model profile (HMM) searches (http://hmmer.org/) performed against the manually curated Pfam-A database (cutoff E value, 1 × 10−10). Furthermore, tRNA genes were identified using tRNAscan-SE version 1.4 (41), while rRNA genes were detected using RNAmmer version 1.2 (42). In order to ensure the consistency of the genomic analyses, B. pseudolongum chromosomes retrieved from public databases were reannotated using the same bioinformatics pipeline applied for the 51 B. pseudolongum strains isolated in the current study.

Comparative genomics.

The genomes of the decoded B. pseudolongum strains (Table 1) were subjected to a pangenome calculation using the PGAP (43). Each predicted proteome of a given B. pseudolongum strain was screened for orthologues against the proteome of every collected B. pseudolongum strain by means of BLAST analysis (44) (cutoff E value, <1 × 10−5; 50% identity over at least 80% of both protein sequences). The resulting output was then clustered into protein families named as Clusters of Orthologous Groups (COGs) by means of MCL (graph theory-based Markov clustering algorithm) (45), using the gene family (GF) method. Pangenome profiles were built using an optimized algorithm incorporated in the PGAP software, based on a presence/absence matrix that included all identified COGs in the 60 analyzed genomes. Protein families that are shared among all analyzed genomes allowed us to formulate the core genome of the B. pseudolongum species. In addition, unique protein families encoded by the analyzed B. pseudolongum genomes that are not present in other genomes were also identified.

Phylogenomic analyses.

The concatenated core genome sequences of each B. pseudolongum strain were aligned using MAFFT software (46), and corresponding phylogenetic trees were constructed using the neighbor-joining method in ClustalW version 2.1 (47). Accordingly, a B. pseudolongum supertree was built using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). For each genome pair, an ANI value was calculated using the program JSpecies, version 1.2.1 (28).

Genomic analyses.

Carbohydrate-active enzymes predicted to be encoded by each of the 60 B. pseudolongum genomes were identified based on similarity to genes incorporated within the Carbohydrate-Active enZyme (CAZy) database (48). For this purpose, we used GH data of 12,039 bacterial genomes available in the CAZy database, retrieving functional annotation by means of RAPSearch2 (cutoff E value, 1 × 10−30). The prediction of genes encoding restriction enzymes was performed by means of the REBASE database (49). Identification of clustered regularly interspaced short palindromic repeats (CRISPRs) was achieved through CRISPRfinder (50), while related Cas-encoding genes were manually evaluated in each B. pseudolongum genome. Identification of genes that are predicted to be acquired by horizontal gene transfer (HGT) events was performed using COLOMBO version 4.0 (51). Prediction of prophage sequences was evaluated by means of a custom database composed of bifidobacterial genes previously classified as prophage genes (29). Additional analyses were performed to detect similarity between CRISPR spacers and prophage sequences retrieved within B. pseudolongum genomes. Functional classification of the B. pseudolongum proteome was performed on the basis of the eggNOG database (21) by means of RAPSearch2 (cutoff E value, 1 × 10−30).

Carbohydrate growth assays.

B. pseudolongum growth on semisynthetic MRS medium supplemented with 1% (wt/vol) of a specific sugar was monitored by optical density at 600 nm using a plate reader (BioTek, Winooski, VT, USA). The plate reader was run for 24 h and 48 h, and readings were preceded by 30 s shaking at medium speed. Cultures were grown in biologically independent triplicates, and the resulting growth data were expressed as the mean of these replicates. Carbohydrates were purchased from Sigma and Carbosynth (Berkshire, UK).

Statistical analyses.

SPSS software (IBM, Italy) was used to perform statistical analysis between strains of the B. pseudolongum subsp. pseudolongum group and B. pseudolongum subsp. globosum group by Student’s t test.

Data availability.

Fifty-one newly isolated B. pseudolongum genomes were sequenced and deposited at DDBJ/ENA/GenBank under the accession numbers reported in Table 1 (BioProject no. PRJNA510800), together representing a collection of 60 B. pseudolongum genomes.

Supplementary Material

Supplemental file 1
AEM.03065-18-s0001.pdf (557.1KB, pdf)
Supplemental file 2
AEM.03065-18-sd002.xlsx (53.9KB, xlsx)

ACKNOWLEDGMENTS

We thank GenProbio srl for financial support of the Laboratory of Probiogenomics. This work was funded by the EU Joint Programming Initiative “A healthy diet for a healthy life” (JPI HDHL; http://www.healthydietforhealthylife.eu/) to D.V.S. (in conjunction with Science Foundation Ireland [SFI], grant 15/JP-HDHL/3280) and to M.V. (in conjunction with MIUR, Italy). D.V.S. is a member of APC Microbiome Ireland funded by the Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (grant SFI/12/RC/2273).

Part of this research was conducted using the High Performance Computing (HPC) facility of the University of Parma.

We declare no conflicts of interest.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.03065-18.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1
AEM.03065-18-s0001.pdf (557.1KB, pdf)
Supplemental file 2
AEM.03065-18-sd002.xlsx (53.9KB, xlsx)

Data Availability Statement

Fifty-one newly isolated B. pseudolongum genomes were sequenced and deposited at DDBJ/ENA/GenBank under the accession numbers reported in Table 1 (BioProject no. PRJNA510800), together representing a collection of 60 B. pseudolongum genomes.

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

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