Analysis of Predicted Carbohydrate Transport Systems Encoded by Bifidobacterium bifidum PRL2010

Evaluation of the growth profile of B. bifidum on different carbohydrates.Bifidobacterial strains used in this study were routinely cultivated in an anaerobic atmosphere (2.99% H₂, 17.01% CO₂, and 80% N₂) in a chamber (Concept 400; Ruskin) at 37°C for 32 h in de Man-Rogosa-Sharpe (MRS) (Scharlau Chemie, Barcelona, Spain) medium supplemented with 0.05% (wt/vol) l-cysteine hydrochloride. Growth curves of bifidobacteria, performed in triplicate, were obtained from cultures grown on a semisynthetic medium supplemented with various carbon sources, which included mono-, oligo-, and polysaccharides. Growth of B. bifidum PRL2010 was examined on 32 different carbohydrates, including 20 carbon sources that had previously been tested (24). As shown in Table 1, B. bifidum PRL2010 is capable of growth on a variety of carbohydrates, which include various disaccharides, such as lactose, and amino sugars, such as N-acetylgalactosamine, as previously shown by Turroni et al. (24). No appreciable growth (i.e., where the final optical density at 600 nm [OD₆₀₀] was below 0.1) was observed on other sugars, such as mannitol, xylan, or α-cyclodextrin. Notably, the obtained carbohydrate utilization profile demonstrates growth on components of host-derived glycans, such as human milk oligosaccharides (HMOs) or mucin, as represented by lactose, galactose, N-acetylgalactosamine, and N-acetylglucosamine. These findings may thus be a direct reflection of the ecological niche in which this microorganism predominates, i.e., the infant colon. However, appreciable growth of B. bifidum PRL2010 (i.e., a final OD₆₀₀ of >0.6) was also noticed on certain plant-derived carbohydrates such as (iso)maltose, melibiose, sucrose, fructo oligosaccharides (FOS), (iso)maltulose, ribose, fructose, and turanose (Table 1). Compared to other bifidobacteria tested or data available in literature (13), and with the exception of Bifidobacterium animalis subsp. lactis BB12, it seems that B. bifidum PRL2010 is able to metabolize a somewhat lower number of carbohydrates (only eight) that sustain reasonable good growth (i.e., a final OD₆₀₀ of >1.2) (Table 1). However, these data require careful interpretation, as they represent a modest selection of carbohydrates and bifidobacterial strains, and therefore, no solid conclusions can be drawn from this.

Genomics of carbohydrate transport systems.A genome sequence survey of B. bifidum PRL2010 employing Artemis software (18) for the presence of genes that encode predicted carbohydrate transporters returned a substantial number of such genes, although this number was lower than that obtained from genome searches of other human enteric bifidobacteria, such as Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium breve (6, 12, 21). In fact, according to the Transporter Classification Database (TCDB; www.tcdb.org), just 25 genes (Fig. 1) are predicted to encode components of transport systems in the B. bifidum PRL2010 genome and dedicated to carbohydrate uptake, whereas the other human intestinal bifidobacterial genomes are predicted to contain between 35 and 68 such genes, as represented by B. adolescentis ATCC 15703 and B. longum subsp. infantis ATCC 15697, respectively (see Table S1 in the supplemental material). B. animalis subsp. lactis BB12 contains just 21 transporter genes, and this rather low number may have been due to genome simplification as a consequence of the continued cultivation of this strain in rather simple substrates during industrial processing. A similar scenario has been noticed for other strains used in the food industry (30).

• Open in new tab
• Download powerpoint

Fig 1

Genetic maps of the predicted carbohydrate transporter gene clusters identified in the genome of B. bifidum PRL2010. Each individual gene is represented by an arrow and is colored or marked according to the predicted function as indicated in the figure. HAD, haloacid dehalogenase.

When the analysis was extended to a nonenteric bifidobacterial strain, such as the oral cavity inhabitant Bifidobacterium dentium Bd1, the relatively low number of identified carbohydrate transporters of B. bifidum PRL2010 is even more obvious (see Table S1 in the supplemental material). The 25 predicted carbohydrate transport proteins encoded by the PRL2010 genome can be classified into different groups: 8 belong to members of the ABC-type family, 12 are similar to various components of PEP-PTS systems, 1 is deduced to be a major intrinsic protein (MIP), while 4 represent secondary carriers, encompassing 2 members of the major facilitator superfamily (MFS), 1 member of the glycoside-pentoside-hexuronide (GPH):cation symporter family, and finally 1 member of the glucose/ribose porter family (GRP). The genes coding for the components of a given ABC transporter for a carbohydrate are commonly organized in a locus, typically encompassing between two and five genes that specify a sugar-specific solute binding protein (SBP), one or two permeases, and one or two predicted ATPase proteins, although such ATPases can be shared among different ABC transporters and the corresponding gene(s) can therefore be absent from such a locus (4). We identified two loci that encode putative carbohydrate uptake ABC-type systems (represented by BBPR_1056 to BBPR_1058, BBPR_1353-BBPR_1354, and BBPR_1356), made up of genes encoding three SBPs (BBPR_1353, BBPR_1354, and BBPR_1058) and three permeases (BBPR_1056, BBPR_1057, and BBPR_1356). Hydropathy profile analysis using the ExPaSy Proteomic Server predicts that PRL2010-encoded permease proteins and SBPs contain six transmembrane domains and one transmembrane domain, respectively (see Fig. S1 in the supplemental material), which is typical for permease and SBP proteins of Gram-positive bacteria (4). Furthermore, the genome of PRL2010 contains a single predicted ATPase-encoding gene (BBPR_1824) as well as an additional SBP-encoding gene (BBPR_1074), neither of which is located next to other genes that specify one or more components of an ABC family carbohydrate uptake system (1, 13). We found a similarity above 60% at the amino acid level (extended to key amino acid residues sustaining enzymatic activities) between the deduced product of BBPR_1824 and that of BL0673 of B. longum subsp. longum NCC2705 and MsiK of Streptomyces coelicolor A3, both of which are believed to encode a universal ATPase for ABC-type carbohydrate transporters identified in these microorganisms (1, 13). These findings suggest that the functionality of the two identified PRL2010 ABC-type carbohydrate uptake systems rely on this single predicted ATPase-encoding gene (Fig. 1). Based on BLAST analysis, the four putative secondary transporters that belong to the MFS, GPH, and GRP families, including proton facilitators and symporters, are expected to transport mono- and disaccharides (Table 2). All these secondary transporters are predicted to consist of a single integral membrane-associated protein, which traverses the membrane 10 to 12 times (Fig. S1).

The genome of PRL2010 also contains four complete PTS systems, represented by the general components histidine protein (HPr) and enzyme I (EI), and four sets of the variable components EIIA, EIIB, and EIIC (see Fig. S1 in the supplemental material).

The organization and location of the carbohydrate transporter-encoding genes identified in the genome of B. bifidum PRL2010 and their closest homologs from all other publicly available bifidobacterial genomes are schematically represented in Fig. 2, where the deduced amino acid sequences of the PRL2010 carbohydrate transporter loci are aligned with homologs of other bifidobacteria. This comparative analysis revealed a high level of conservation (>70% identity) between the ABC permeases, SBP, and some of the PTS component proteins found in various bifidobacteria. In contrast, the EIIA, EIIB, and EIIC enzymes encoded by BBPR_1715, BBPR_1716, and BBPR_1717, respectively, and predicted to specify a complete PTS system, and BBPR_0366, which encodes a membrane-embedded EIIC carbohydrate transport component, only appeared to be present in the genomes of strains of B. bifidum, thus apparently representing unique genetic features of this species and supporting the notion that such transporters may provide a specific phenotype or particular niche access to B. bifidum strains.

• Open in new tab
• Download powerpoint

Fig 2

Schematic comparative representation of carbohydrate transporter-encoding genes of B. bifidum PRL2010 and of various other bifidobacterial strains such as B. dentium Bd1, B. adolescentis ATCC 15703, B. breve UCC2003, B. longum subsp. longum NCC2705, B. longum subsp. longum DJO10A, B. longum subsp. infantis ATCC 15697, and B. animalis subsp. lactis BB12. Each arrow indicates an open reading frame (ORF), the size of which is proportional to the length of the arrow. Predicted protein function is indicated above each arrow. The corresponding amino acid identity as a percentage is indicated.

Notably, many of the gene clusters involved in carbohydrate transport contain genes encoding putative regulators, which may indicate that such gene clusters are subject to substrate-dependent regulation in a similar fashion as previously shown for other bifidobacterial carbohydrate metabolism loci (14, 23). The genes specifying putative ABC-type permeases as well as PTS systems were indeed flanked by genes encoding such predicted regulators such as carbohydrate kinases belonging to the ROK (repressor, open reading frame, kinase) family (BBPR_1053-BBPR_1054), a transcription antiterminator of the BglG type (BBPR_1507), a DeoR-type regulator (BBPR_0365), and LacI-type regulators (BBPR_0029 and BBPR_1734) (Fig. 1). Similarly, the predicted MFS- and GPH-encoding genes present in the PRL2010 genome are located close to genes specifying putative regulators of the LacI type (BBPR_0143 and BBPR_1462) or belonging to the ROK family (BBPR_0565) (Fig. 1).

Phylogenetic analysis based on SBP protein sequences.In order to assess the distribution of SBP homologs across the Bifidobacterium genus, we surveyed available bifidobacterial genomic data for such genes, whose protein products were then aligned using ClustalW to produce an unrooted neighbor-joining phylogenetic tree, which was built using PHYLIP (Phylogeny Inference Package, version 3.5c) (5) (see Fig. S2 in the supplemental material). All analyzed SBPs, except for the B. bifidum PRL2010 SBP encoded by BBPR_1074, were shown to be closely related to members of the families Sbp_bac_1 and Sbp_bac_3 (22). Thus, BBPR_1074 might encode a novel solute binding protein never identified previously in bifidobacteria and consequently suggesting the existence of a novel family of SBP.

Transcriptional analysis of carbohydrate transporter loci.In order to determine whether the genes encoding the predicted carbohydrate transporters, including components of the ABC systems, PTS systems, and secondary carriers (MFS, GPH, and GRP), as identified on the B. bifidum PRL2010 genome are differentially transcribed when PRL2010 is cultivated on different carbohydrate growth substrates, we evaluated the level of predicted transporter gene-specific mRNAs by quantitative real-time PCR (RT-qPCR) assays according to the guidelines of Bustin et al. (2). Such experiments were performed using mRNA samples extracted from three independent exponentially growing cultures of B. bifidum PRL2010, which had been resuspended in prewarmed MRS medium containing one of a varied set of carbohydrates. Aliquots of 20 ml of PRL2010 cultures were centrifuged for 10 min at 4,000 × g at 4°C in the presence of RNA-later (Ambion). The pellets were then immediately frozen in liquid nitrogen and submitted to RNA extraction using a previously described method, which includes a DNase treatment (35). The quality and integrity of the RNA were checked by Experion (Bio-Rad) analysis. cDNA was synthesized and purified using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA), according to the supplier's instructions. RT-qPCR primers used including those for normalization are described in Table 3. Primers used for normalization were those previously described (25). Criteria for primer design were a desired melting temperature (T_m) value between 58 and 60°C and an amplicon size of approximately 100 to 200 bp. RT-qPCR was performed using the CFX96 system (Bio-Rad, Hercules, CA). PCR products were detected with SYBR green fluorescent dye and amplified according to the following protocol: one cycle of 95°C for 3 min, followed by 39 cycles, with 1 cycle consisting of 95°C for 5 s and 66°C for 20 s. The melting curve was measured from 65°C to 95°C with increments of 0.5°C/s.

Each PCR mix contained 12.5 μl of 2× SYBR SuperMix Green (Bio-Rad, Hercules, CA), 1 μl of cDNA dilution, each of the forward and reverse primers at 0.5 μM, and nuclease-free water to obtain a final volume of 20 μl. In each run, a negative control (no cDNA) for each primer set was included. The expression ratio of the selected genes was calculated and analyzed using the CFX Manager expression software (Bio-Rad, Hercules, CA). The cutoff value applied to highlight significant change in the expression was 4.

The mRNA levels corresponding to genes encoding individual components of identified B. bifidum PRL2010 carbohydrate transporters were shown to be variable in response to the various carbon sources used for growth (Fig. 3). Interestingly, the transcription of three predicted carbohydrate transport systems of PRL2010, i.e., the ABC locus (BBPR_1353 to BBPR_1356) (this presumed ABC-type transporter also exhibits induction upon growth in turanose [see below]) and two PTS loci (BBPR_0032 and BBPR_0240), was significantly enhanced when PRL2010 was cultivated on host-specific glycans such as mucin and host glycan constituents like N-acetylglucosamine (Fig. 3), and this may reflect a specialization of PRL2010 toward the particular ecological niche of the human GIT.

• Open in new tab
• Download powerpoint

Fig 3

Relative transcription levels of carbohydrate transporter-encoding genes from B. bifidum PRL2010 upon cultivation in MRS medium supplemented with various carbon sources as analyzed by quantitative real-time PCR assays. The histograms indicate the relative amounts of the carbohydrate transporter-encoding gene mRNAs for the specific samples. The y axis indicates the fold induction of the investigated gene compared to the reference condition (lactose, which is indicated by §, or glucose, which is indicated by •). The x axis represents the different carbohydrates tested for each ORF. In each panel, the ORF numbering indicates the gene code according to Fig. 1. Only those genes/carbohydrate conditions whose expression was significantly changed are shown. An asterisk on the y axis indicates that the change in fold induction is considered significant. The error bar for each column represent the standard deviation calculated from three replicates. GOS, galacto oligosaccharides.

Furthermore, we identified genes that represent predicted carbohydrate transporter components and that were shown to exhibit specific transcriptional induction as a result of growth on particular monosaccharides such as fructose (BBPR_0561), glucose (BBPR_1508), ribose (BBPR_0366 and BBPR_1399), galactose (BBPR_1459) or disaccharides such as the sucrose-like carbohydrate turanose (BBPR_0145 and BBPR_1715, BBPR_1717, and BBPR_1353 to BBPR_1356) (Fig. 4). Each of the genes that specify one of the ABC-type carbohydrate transporters, specified by BBPR_1056 to BBPR_1058, as well as the gene encoding SBP_{BBPR_1074} were shown to be induced when B. bifidum PRL2010 was grown on multiple carbon sources, reminiscent of what previously was described for several ABC systems identified in the genome of B. longum subsp. longum NCC2705 (8, 13). These data suggest that transcription of these transporters is subject to a common control mechanism and thus not necessarily regulated in response to their specific substrate.

• Open in new tab
• Download powerpoint

Fig 4

Identification of the conservation of carbohydrate transporter genes within several members of the B. bifidum taxon. The presence (black) or absence (gray) of key genes predicted to be involved in carbohydrate transporters in the currently publicly available B. bifidum genomes and on the basis of CGH data (24) is shown.

Furthermore, relative expression levels between genes that are predicted to encode components of a particular carbohydrate transporter varied quite substantially when B. bifidum PRL2010 was grown on a specific carbohydrate. This finding is not unusual for SBP-encoding genes, because higher transcription is expected to lead to an abundance of SBP molecules (13), thus providing a more efficient substrate scavenging ability and consequent uptake. Since the SBPs of strain PRL2010 are encoded by distinct genes, we may envisage that multiple protein copies of SBP are cooperating with a corresponding carbohydrate permease, in a similar fashion to that described for other bacteria (3, 13, 15). The observation that a range of substrates can induce the expression of predicted SBP-encoding genes in B. bifidum PRL2010 may be linked to their low number in this genome with respect to other bifidobacterial genomes (e.g., 16 in B. breve UCC2003, 11 in B. longum subsp. longum NCC2705 and 20 in B. longum subsp. infantis ATCC 15697) and thus may be pivotal for the colonization strategy of PRL2010. Furthermore, it is tempting to speculate that PRL2010 has specialized itself in the metabolism of a relatively low number of carbohydrates and that the apparently low level of induction specificity of the SBP-encoding genes and other carbohydrate-transporting genes is just a consequence of this adaptation.

Evaluation of genes involved in carbohydrate transport in members of the B. bifidum species.In order to investigate the level of conservation of the genetic repertoire involved in carbohydrate transport within the B. bifidum taxon, we surveyed the currently publicly available B. bifidum genome sequences, i.e., B. bifidum S17 (36) and B. bifidum NCIMB 41171 (GenBank accession no. NZ_ABQP00000000) as well as previously published comparative genomic hybridization (CGH) data (24). The obtained data demonstrated that a large proportion of the 25 identified PRL2010 carbohydrate transport genes are conserved within these investigated members of the B. bifidum taxon. Only BBPR_1056 and BBPR_1508 were found to be variably present in the tested B. bifidum strains (Fig. 4). Comparative analyses highlighted a high similarity (higher than 98% at nucleotide level) of these transporter genes with that identified in the genome of B. bifidum PRL2010, thus corroborating the CGH findings (Fig. 4).