The Bifidobacterium dentium Bd1 genome sequence reflects its genetic adaptation to the human oral cavity

Abstract

Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.

Figure 1. Circular genome map of B. dentium Bd1.

From innermost circle, circle (1) illustrates GC skew (G−C/G+C), values >0 are in red and <0 in green. Circle (2) highlights G+C% deviation from the mean (58.54%). Circle (3) indicates rRNAs (depicted in red) and tRNAs (depicted in blue). Circle (4) denotes IS and prophages (depicted in orange). Circle (5) depicts genes involved in sugar metabolism according to the CAZY database. Circle (6) denotes conserved ORF distribution. Circle (7) shows coding regions by strand with color corresponding to the COG functional assignment (the color code used is the same indicated in Figure 2). Circle (8) displays the ORF distribution by strand.

Figure 2. Comparison of COG functional categories between completely sequenced bifidobacterial genomes.

Each coloured segment indicates the relative contribution of a functional category as a percentage of total COGs. Each ring indicates a different bifidobacterial genome as labelled. The color of each COG family is indicated in the Figure The name of the bacterial genomes are indicated in the Figure.

Figure 3. Functional annotation and assignment of the encoded proteins from the genomes of B. dentium Bd1 and B. longum subsp. longum NCC2705 in different superfamily categories according to the Fugue fold recognition method.

In (A), each bar represents the odd ratio between B. dentium Bd1 and B. longum subsp. longum NCC2705. The bar corresponding to the toxins/defence superfamily is indicated. In the y-axis is indicated the value of the odd ratio between B. dentium Bd1 and B. longum subsp. longum NCC2705. (B) indicates the different B. dentium Bd1 ORFs classified in the toxins/defence superfamily.

Figure 4. Dot plot of B. dentium Bd1 versus B. dentium ATCC27678.

The visible areas of divergence are indicated. Blue type indicates sequences unique to Bd1 compared with ATCC27678, whereas red type shown sequences absent in Bd1. The deduced functions of the divergent regions are described in the table.

Figure 5. Mobile genetic elements of the B. dentium Bd1 genome.

IS elements and predicted prophage-like elements are labelled in red and green, respectively. The first plot from the bottom indicates the deviation of the G+C content of each ORF of the B. dentium Bd1 genome from the mean average (58.54%). In the second plot each dot represents an ORF displaying a biased codon usage determined by factorial correspondence analysis of codon usage.

Figure 6. Genomic diversity in the B. dentium species with reference to the B. dentium strain Bd1 genome (Left) CGH data.

Each horizontal row corresponds to a probe on the array, and genes are ordered vertically according to their position on the Bd1 genome. The columns represent the analysed strains, and strains are identified by their code numbers. The colour code corresponding to the presence/absence is given at the top right of the figure: the gradient goes from black to green to indicate the presence, divergence or absence of a gene sequence. The predicted function of some relevant genes are shown on the right-hand margin, ori: origin of replication; ter, terminus of replication (left-hand inset). Right-hand inset displays Signal ratio distribution of the CGH data. The reference is B. dentium strain Bd1. Ratios are expressed in a log₂ scale. See text for details.

Figure 7. Analysis of the glycobiome of the B. dentium Bd1 genome by reference to CAZy database.

(A) The glycoside-hydrolase (GH) families identified in the genome of B. dentium Bd1 and in enteric bifidobacterial genomes. (B) The glycosyl-transferase families (GT) encoded by the genome of B. dentium Bd1 and by enteric bifidobacterial genomes. (C) The GH families identified in the genome of B. dentium Bd1 and in other oral pathogens. (D) The GT families identified in the genome of B. dentium Bd1 and in other oral pathogens. In each panel, the X-axis represents the different GH families or GT families according to the CAZy database (Henrissat 1999), whereas the Y-axis indicates the abundance of each GH family expressed in percentage.

Figure 8. Identification of B. dentium Bd1 transcribed genes by DNA–micro array analysis.

The heat-map indicates the change in the expression upon cultivation of Bd1 cells at low pH as well as represents selected genes that were up- or down-regulated when grown in media containing various carbohydrates as the sole carbon sources as compared to growth on glucose. Each row represents a separate transcript and each column represents a separate sample. Colour legend is on the top of each micro array plot, red indicates increased transcription levels, whereas green indicates decreased trasncription level as compared to the reference samples (glucose-grown samples or growth at pH 6).

Figure 9. Comparison of presumptive fimbrial loci in B. dentium Bd1 with the corresponding loci in different oral bacteria.

Each arrow indicates an ORF. The length of the arrow is proportional to the length of the predicted open reading frame. Corresponding genes are marked with the same colour. The putative function of the protein is indicated above each arrow.