Bifidobacterial Genomes Research Articles

Structure of co-expression networks of Bifidobacterium species in response to human milk oligosaccharides.

Biological systems respond to environmental perturbations and a large diversity of compounds through gene interactions, and these genetic factors comprise complex networks. Experimental information from transcriptomic studies has allowed the identification of gene networks that contribute to our understanding of microbial adaptations. In this study, we analyzed the gene co-expression networks of three Bifidobacterium species in response to different types of human milk oligosaccharides (HMO) using weighted gene co-expression analysis (WGCNA). RNA-seq data obtained from Geo Datasets were obtained for Bifidobacterium longum subsp. Infantis, Bifidobacterium bifidum and Bifidobacterium longum subsp. Longum. Between 10 and 20 co-expressing modules were obtained for each dataset. HMO-associated genes appeared in the modules with more genes for B. infantis and B. bifidum, in contrast with B. longum. Hub genes were identified in each module, and in general they participated in conserved essential processes. Certain modules were differentially enriched with LacI-like transcription factors, and others with certain metabolic pathways such as the biosynthesis of secondary metabolites. The three Bifidobacterium transcriptomes showed distinct regulation patterns for HMO utilization. HMO-associated genes in B. infantis co-expressed in two modules according to their participation in galactose or N-Acetylglucosamine utilization. Instead, B. bifidum showed a less structured co-expression of genes participating in HMO utilization. Finally, this category of genes in B. longum clustered in a small module, indicating a lack of co-expression with main cell processes and suggesting a recent acquisition. This study highlights distinct co-expression architectures in these bifidobacterial genomes during HMO consumption, and contributes to understanding gene regulation and co-expression in these species of the gut microbiome.

Genome-Wide Assessment of Stress-Associated Genes in Bifidobacteria.

ABSTRACTOver the last decade, the genomes of several Bifidobacterium strains have been sequenced, delivering valuable insights into their genetic makeup. However, bifidobacterial genomes have not yet been systematically mined for genes associated with stress response functions and their regulation. In this work, a list of 76 genes related to stress response in bifidobacteria was compiled from previous studies. The prevalence of the genes was evaluated among the genome sequences of 171 Bifidobacterium strains. Although genes of the protein quality control and DNA repair systems appeared to be highly conserved, genome-wide in silico screening for consensus sequences of putative regulators suggested that the regulation of these systems differs among phylogenetic groups. Homologs of multiple oxidative stress-associated genes are shared across species, albeit at low sequence similarity. Bee isolates were confirmed to harbor unique genetic features linked to oxygen tolerance. Moreover, most studied Bifidobacterium adolescentis and all Bifidobacterium angulatum strains lacked a set of reactive oxygen species-detoxifying enzymes, which might explain their high sensitivity to oxygen. Furthermore, the presence of some putative transcriptional regulators of stress responses was found to vary across species and strains, indicating that different regulation strategies of stress-associated gene transcription contribute to the diverse stress tolerance. The presented stress response gene profiles of Bifidobacterium strains provide a valuable knowledge base for guiding future studies by enabling hypothesis generation and the identification of key genes for further analyses.IMPORTANCE Bifidobacteria are Gram-positive bacteria that naturally inhabit diverse ecological niches, including the gastrointestinal tract of humans and animals. Strains of the genus Bifidobacterium are widely used as probiotics, since they have been associated with health benefits. In the course of their production and administration, probiotic bifidobacteria are exposed to several stressors that can challenge their survival. The stress tolerance of probiotic bifidobacteria is, therefore, an important selection criterion for their commercial application, since strains must maintain their viability to exert their beneficial health effects. As the ability to cope with stressors varies among Bifidobacterium strains, comprehensive understanding of the underlying stress physiology is required for enabling knowledge-driven strain selection and optimization of industrial-scale production processes.

Diversity of Human-Associated Bifidobacterial Prophage Sequences.

Members of Bifidobacterium play an important role in the development of the immature gut and are associated with positive long-term health outcomes for their human host. It has previously been shown that intestinal bacteriophages are detected within hours of birth, and that induced prophages constitute a significant source of such gut phages. The gut phageome can be vertically transmitted from mother to newborn and is believed to exert considerable selective pressure on target prokaryotic hosts affecting abundance levels, microbiota composition, and host characteristics. The objective of the current study was to investigate prophage-like elements and predicted CRISPR-Cas viral immune systems present in publicly available, human-associated Bifidobacterium genomes. Analysis of 585 fully sequenced bifidobacterial genomes identified 480 prophage-like elements with an occurrence of 0.82 prophages per genome. Interestingly, we also detected the presence of very similar bifidobacterial prophages and corresponding CRISPR spacers across different strains and species, thus providing an initial exploration of the human-associated bifidobacterial phageome. Our analyses show that closely related and likely functional prophages are commonly present across four different species of human-associated Bifidobacterium. Further comparative analysis of the CRISPR-Cas spacer arrays against the predicted prophages provided evidence of historical interactions between prophages and different strains at an intra- and inter-species level. Clear evidence of CRISPR-Cas acquired immunity against infection by bifidobacterial prophages across several bifidobacterial strains and species was obtained. Notably, a spacer representing a putative major capsid head protein was found on different genomes representing multiple strains across B. adolescentis, B. breve, and B. bifidum, suggesting that this gene is a preferred target to provide bifidobacterial phage immunity.

Assembly, Annotation, and Comparative Analysis of Bifidobacterial Genomes.

Genome assembly and annotation are two of the key actions that must be undertaken in order to explore the genomic repertoire of (bifido)bacteria. The gathered information can be employed to genomically characterize a given microorganism, and can also be used to perform comparative genome analysis by including other sequenced (bifido)bacterial strains. Here, we highlight various bioinformatic programs able to manage next generation sequencing data starting from the assembly of a genome to the comparative analyses between strains.

Literature-Based Phenotype Survey and In Silico Genotype Investigation of Antibiotic Resistance in the Genus Bifidobacterium

Bifidobacteria are typical commensals inhabiting the human intestine and are beneficial to the host because of their probiotic properties. One of the risks concerning probiotics is the potential of introducing antibiotic resistance genes (ARGs) to the host gut pathogens. This study was aimed to depict the general antibiotic resistance characteristics of the genus Bifidobacterium by combining the reported phenotype dataset and in silico genotype prediction. Bifidobacteria were mostly reported to be sensitive to beta-lactams, glycopeptides, chloramphenicol, and rifampicin, but resistant to aminoglycosides, polypeptides, quinolones, and mupirocin. Generally, the resistance phenotypes to erythromycin, tetracycline, fusidic acid, metronidazole, clindamycin, and trimethoprim were variable. Besides cmX and tetQ, characterized in bifidobacterial resident plasmids, 3520 putative ARGs were identified from 831 bifidobacterial genomes through BLASTP search. The identified ARGs matched thirty-eight reference ARGs, four of which seemed to be mutant housekeeping genes. The two high-abundant ARGs, tetW and ermX, were found to have different distribution traits. The predicted ARGs reasonably explained most of the corresponding resistant phenotypes in the published literature.

Mobilome and Resistome Reconstruction from Genomes Belonging to Members of the Bifidobacterium Genus.

Specific members of the genus Bifidobacterium are among the first colonizers of the human/animal gut, where they act as important intestinal commensals associated with host health. As part of the gut microbiota, bifidobacteria may be exposed to antibiotics, used in particular for intrapartum prophylaxis, especially to prevent Streptococcus infections, or in the very early stages of life after the birth. In the current study, we reconstructed the in silico resistome of the Bifidobacterium genus, analyzing a database composed of 625 bifidobacterial genomes, including partial assembled strains with less than 100 genomic sequences. Furthermore, we screened bifidobacterial genomes for mobile genetic elements, such as transposases and prophage-like elements, in order to investigate the correlation between the bifido-mobilome and the bifido-resistome, also identifying genetic insertion hotspots that appear to be prone to horizontal gene transfer (HGT) events. These insertion hotspots were shown to be widely distributed among analyzed bifidobacterial genomes, and suggest the acquisition of antibiotic resistance genes through HGT events. These data were further corroborated by growth experiments directed to evaluate bacitracin A resistance in Bifidobacterium spp., a property that was predicted by in silico analyses to be part of the HGT-acquired resistome.

Exploration of Survival Traits, Probiotic Determinants, Host Interactions, and Functional Evolution of Bifidobacterial Genomes Using Comparative Genomics.

Members of the genus Bifidobacterium are found in a wide-range of habitats and are used as important probiotics. Thus, exploration of their functional traits at the genus level is of utmost significance. Besides, this genus has been demonstrated to exhibit an open pan-genome based on the limited number of genomes used in earlier studies. However, the number of genomes is a crucial factor for pan-genome calculations. We have analyzed the pan-genome of a comparatively larger dataset of 215 members of the genus Bifidobacterium belonging to different habitats, which revealed an open nature. The pan-genome for the 56 probiotic and human-gut strains of this genus, was also found to be open. The accessory- and unique-components of this pan-genome were found to be under the operation of Darwinian selection pressure. Further, their genome-size variation was predicted to be attributed to the abundance of certain functions carried by genomic islands, which are facilitated by insertion elements and prophages. In silico functional and host-microbe interaction analyses of their core-genome revealed significant genomic factors for niche-specific adaptations and probiotic traits. The core survival traits include stress tolerance, biofilm formation, nutrient transport, and Sec-secretion system, whereas the core probiotic traits are imparted by the factors involved in carbohydrate- and protein-metabolism and host-immunomodulations.

Reconstruction of the Bifidobacterial Pan-Secretome Reveals the Network of Extracellular Interactions between Bifidobacteria and the Infant Gut.

The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant, as they regulate the interaction with the gut environment. In the current study, we screened the predicted proteome of over 300 bifidobacterial strains among the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze human milk oligosaccharides (HMOs) and intestinal glycoconjugates, such as mucin. Furthermore, an exploration of metatranscriptomic data sets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut.IMPORTANCE The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria is incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of the secretion systems between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii.

Impact of gut-associated bifidobacteria and their phages on health: two sides of the same coin?

Bifidobacteria are among the first microbial colonisers of the human infant gut post-partum. Their early appearance and dominance in the human infant gut and the reported health-promoting or probiotic status of several bifidobacterial strains has culminated in intensive research efforts that focus on their activities as part of the gut microbiota and the concomitant implications for human health. In this mini-review, we evaluate current knowledge on the genomics of this diverse bacterial genus, and on the genetic and functional adaptations that have underpinned the success of bifidobacteria in colonising the infant gut. The growing interest in functional genomics of bifidobacteria has also created interest in the interactions of bifidobacteria and their (bacterio)phages. While virulent phages of bifidobacteria have yet to be isolated, the incidence of integrated (pro)phages in bifidobacterial genomes are widely reported and this mini-review considers the role of these so-called bifidoprophages in modulating bifidobacterial populations in the human gastrointestinal tract and the implications for existing and future development of probiotic therapies.

Omics of bifidobacteria: research and insights into their health-promoting activities.

Members of the genus Bifidobacterium include gut commensals that are particularly abundant among the microbial communities residing in the gut of healthy breast-fed infants, where their presence has been linked to many beneficial host effects. Next-generation DNA sequencing and comparative and functional genome methodologies have been shown to be particularly useful in exploring the diversity of this genus. These combined approaches have allowed the identification of genetic features related to bifidobacterial establishment in the gut, involving host-microbe as well as microbe-microbe interactions. Among these, proteinaceous structures, which protrude from the bacterial surface, i.e. pili or fimbriae, and exopolysaccharidic cell surface layers or capsules represent crucial features that assist in their colonization and persistence in the gut. As bifidobacteria are colonizers of the large intestine, they have to be able to cope with various sources of osmotic, oxidative, bile and acid stress during their transit across the gastric barrier and the small intestine. Bifidobacterial genomes thus encode various survival mechanisms, such as molecular chaperones and efflux pumps, to overcome such challenges. Bifidobacteria represent part of an anaerobic gut community, and feed on nondigestible carbohydrates through a specialized fermentative metabolic pathway, which in turn produces growth substrates for other members of the gut community. Conversely, bifidobacteria may also be dependent on other (bifido)bacteria to access host- and diet-derived glycans, and these complex co-operative interactions, based on resource sharing and cross-feeding strategies, represent powerful driving forces that shape gut microbiota composition.

Genome analysis of food-processing stressful-resistant probiotic Bifidobacterium animalis subsp. lactis BF052, and its potential application in fermented soymilk.

In this study, Bifidobacterium animalis subsp. lactis BF052 was demonstrated the growth capability in soymilk and could be thus supplemented as a probiotic starter that employed soymilk as one of its food vehicles. The complete genome sequence of BF052 was therefore determined to understand the genetic basis of BF052 as a technological and functional probiotic starter. The whole genome sequence of BF052 consists of a circular genome of 1938 624 bp with a G+C content of 60.50%. This research highlights relevant genes involving in its adaptive responses to industrial and/or environmental stresses and utilization of α-galacto-oligosaccharides in BF052 strain compared with other representative bifidobacterial genomes.

Modulation of the eps-ome transcription of bifidobacteria through simulation of human intestinal environment.

Bacterial exopolysaccharides (EPSs) are mono- or oligo-saccharide units linked by glycosidic bonds, forming homo- or hetero-polymers. In gut commensals, these macromolecules are claimed to protect bacterial cells against gastrointestinal challenges and to be involved in modulating the cross talk between the producing bacterium and its gut environment. The predicted EPS arsenal of theBifidobacteriumgenus, which we designate here as theeps-ome, consists of 9epsgene clusters conserved among different bifidobacterial species and a further 44 uniqueepsloci, together representing a large proportion of the inter(sub)species variability identified among bifidobacterial genomes. Co-cultivations of bifidobacterial species in media simulating adult and infant human gut environments resulted in an increased transcription of key genes for EPS biosynthesis, including glycosyltransferase-encoding genes, as well as genes specifying EPS transporter and polymerase functions, and saccharide biosynthesis and modification enzymes.

Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut.

Bifidobacteria are common and frequently dominant members of the gut microbiota of many animals, including mammals and insects. Carbohydrates are considered key carbon sources for the gut microbiota, imposing strong selective pressure on the complex microbial consortium of the gut. Despite its importance, the genetic traits that facilitate carbohydrate utilization by gut microbiota members are still poorly characterized. Here, genome analyses of 47 representative Bifidobacterium (sub)species revealed the genes predicted to be required for the degradation and internalization of a wide range of carbohydrates, outnumbering those found in many other gut microbiota members. The glycan-degrading abilities of bifidobacteria are believed to reflect available carbon sources in the mammalian gut. Furthermore, transcriptome profiling of bifidobacterial genomes supported the involvement of various chromosomal loci in glycan metabolism. The widespread occurrence of bifidobacterial saccharolytic features is in line with metagenomic and metatranscriptomic datasets obtained from human adult/infant faecal samples, thereby supporting the notion that bifidobacteria expand the human glycobiome. This study also underscores the hypothesis of saccharidic resource sharing among bifidobacteria through species-specific metabolic specialization and cross feeding, thereby forging trophic relationships between members of the gut microbiota.

Starch and starch hydrolysates are favorable carbon sources for bifidobacteria in the human gut.

BackgroundBifidobacteria are key commensals in human gut, and their abundance is associated with the health of their hosts. Although they are dominant in infant gut, their number becomes lower in adult gut. The changes of the diet are considered to be main reason for this difference. Large amounts of whole-genomic sequence data of bifidobacteria make it possible to elucidate the genetic interpretation of their adaptation to the nutrient environment. Among the nutrients in human gut, starch is a highly fermentable substrate and can exert beneficial effects by increasing bifidobacteria and/or being fermented to short chain fatty acids.ResultsIn order to determine the potential substrate preference of bifidobacteria, we compared the glycoside hydrolase (GH) profiles of a pooled-bifidobacterial genome (PBG) with a representative microbiome (RM) of the human gut. In bifidobacterial genomes, only 15% of GHs contained signal peptides, suggesting their weakness in utilization of complex carbohydrate, such as plant cell wall polysaccharides. However, compared with other intestinal bacteria, bifidobacteiral genomes encoded more GH genes for degrading starch and starch hydrolysates, indicating that they have genetic advantages in utilizing these substrates. Bifidobacterium longum subsp. longum BBMN68 isolated from centenarian’s faeces was used as a model strain to further investigate the carbohydrate utilization. The pathway for degrading starch and starch hydrolysates was the only complete pathway for complex carbohydrates in human gut. It is noteworthy that all of the GH genes for degrading starch and starch hydrolysates in the BBMN68 genome were conserved in all studied bifidobacterial strains. The in silico analyses of BBMN68 were further confirmed by growth experiments, proteomic and real-time quantitative PCR (RT-PCR) analyses.ConclusionsOur results demonstrated that starch and starch hydrolysates were the most universal and favorable carbon sources for bifidobacteria. The low amount of these carbon sources in adult intestine was speculated to contribute to the low relative abundance of bifidobacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0362-3) contains supplementary material, which is available to authorized users.

Genomic encyclopedia of type strains of the genus Bifidobacterium.

Bifidobacteria represent one of the dominant microbial groups that are present in the gut of various animals, being particularly prevalent during the suckling stage of life of humans and other mammals. However, the overall genome structure of this group of microorganisms remains largely unexplored. Here, we sequenced the genomes of 42 representative (sub)species across the Bifidobacterium genus and used this information to explore the overall genetic picture of this bacterial group. Furthermore, the genomic data described here were used to reconstruct the evolutionary development of the Bifidobacterium genus. This reconstruction suggests that its evolution was substantially influenced by genetic adaptations to obtain access to glycans, thereby representing a common and potent evolutionary force in shaping bifidobacterial genomes.

Purification and characterization of oxygen-inducible haem catalase from oxygen-tolerant Bifidobacterium asteroides

Bifidobacterium asteroides, originally isolated from honeybee intestine, was found to grow under 20% O(2) conditions in liquid shaking culture using MRS broth. Catalase activity was detected only in cells that were exposed to O(2) and grown in medium containing a haem source, and these cells showed higher viability on exposure to H(2)O(2). Passage through multiple column chromatography steps enabled purification of the active protein, which was identified as a homologue of haem catalase on the basis of its N-terminal sequence. The enzyme is a homodimer composed of a subunit with a molecular mass of 55 kDa, and the absorption spectrum shows the typical profile of bacterial haem catalase. A gene encoding haem catalase, which has an amino acid sequence coinciding with the N-terminal amino acid sequence of the purified protein, was found in the draft genome sequence data of B. asteroides. Expression of the katA gene was induced in response to O(2) exposure. The haem catalase from B. asteroides shows about 70-80% identity with those from lactobacilli and other lactic acid bacteria, and no homologues were found in other bifidobacterial genomes.

Comparative genomics of the genus Bifidobacterium

Whole-genome sequencing efforts have revolutionized the study of bifidobacterial genetics and physiology. Unfortunately, the sequence of a single genome does not provide information on bifidobacterial genetic diversity and on how genetic variability supports improved adaptation of these bacteria to the environment of the human gastrointestinal tract (GIT). Analysis of nine genomes from bifidobacterial species showed that such genomes display an open pan-genome structure. Mathematical extrapolation of the data indicates that the genome reservoir available to the bifidobacterial pan-genome consists of more than 5000 genes, many of which are uncharacterized, but which are probably important to provide adaptive abilities pertinent to the human GIT. We also define a core bifidobacterial gene set which will undoubtedly provide a new baseline from which one can examine the evolution of bifidobacteria. Phylogenetic investigation performed on a total of 506 orthologues that are common to nine complete bifidobacterial genomes allowed the construction of a Bifidobacterium supertree which is largely concordant with the phylogenetic tree obtained using 16S rRNA genes. Moreover, this supertree provided a more robust phylogenetic resolution than the 16S rRNA gene-based analysis. This comparative study of the genus Bifidobacterium thus presents a foundation for future functional analyses of this important group of GIT bacteria.

Analyses of bifidobacterial prophage-like sequences

The genomes of 22 putative prophages (bifidoprophages), previously identified in bifidobacterial genomes, were analyzed to detect the presence and organization of functional modules. Bifidoprophages were shown to display a classical modular genomic organization in which the DNA lysogeny module and the DNA packaging regions are the most highly conserved. Furthermore, single phage gene as well as multiple phage gene-based phylogenetic analyses clearly revealed the chimeric make-up of the genomes of bifidoprophages.

Milk and two oligosaccharides

This month's Genome Watch reviews three recent papers that describe bifidobacterial genomes.

Genome Sequence of the Probiotic Bacterium Bifidobacterium animalis subsp. lactis AD011

Bifidobacterium animalis subsp. lactis is a probiotic bacterium that naturally inhabits the guts of most mammals, including humans. Here we report the complete genome sequence of B. animalis subsp. lactis AD011 that was isolated from an infant fecal sample. Biological functions encoded in a single circular chromosome of 1,933,695 bp, smallest among the completely sequenced bifidobacterial genomes, are suggestive of their probiotic functions, such as utilization of bifidogenic factors and a variety of glycosidic enzymes and biosynthesis of polysaccharides.