Genetic variability of mutans streptococci revealed by wide whole-genome sequencing

Abstract

Background: Mutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood.

Results: Genomes of 6 clinical S. mutans isolates of different origins, one isolate of S. sobrinus (DSM 20742) and one isolate of S. ratti (DSM 20564) were sequenced and comparatively analyzed. Genome alignment revealed a mosaic-like structure of genome arrangement. Genes related to pathogenicity are found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Analysis of genome-scale metabolic networks revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form with other enzymes a novel energetic pathway in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway.Using 67 S. mutans genomes currently available including the strains sequenced in this study, we estimates the theoretical core genome size of S. mutans, and performed modeling of S. mutans pan-genome by applying different fitting models. An "open" pan-genome was inferred.

Conclusions: The comparative genome analyses revealed diversities in the mutans streptococci group, especially with respect to the virulence related genes and metabolic pathways. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them.

Figure 1

Phylogenetic analysis of the 10 mutans streptococci strains compared in this study and their phylogenetic relationship to other Streptococcus species with genomes known before 01/01/2011. a) 16S rRNA phylogenetic tree of Streptococcus species with genomes known before 01/01/2011 (Since the 16S rRNA sequences were almost identical between the different S. mutans strains, only UA159 is shown as representative). b) Phylogenetic tree of mutans streptococci constructed with the core-genome SNPs obtained by PGAP pipeline [11]. All phylogenetic trees were constructed using ClustalX and Phylip by applying the maximum likelihood (ML) method with bootstrap value set to 100. Values beside branches depict ML bootstrap support values. The scale bars in the unit of “substitution/site” are shown below the trees.

Figure 2

Comparison of local collinear blocks (LCBs) of chromosomal sequences of the eight S. mutans strains. In total 16 local LCBs, marked as A to P, were generated and compared by applying the MAUVE software [13,14] with default settings and using strain UA159 as reference. The red vertical bars indicate contig ends. The white areas inside each LCB show regions with low similarities.

Figure 3

Core and pan-genome model of 67 S. mutans genomes. a) Core-genome model of S. mutans. The core-genome size (number of common genes) is plotted as a function of the number (n) of genomes according to a previously proposed model $F_c\left(n\right)={\kappa }_{c}exp⌊\raisebox{1ex}{$-n$}\!\left/ \!\raisebox{-1ex}{${\tau }_c$}\right.⌋+\mathrm{\Omega },$ where K_c, τ_c and Ω are model parameters. Red rectangles are the medians of the core-genome sizes calculated by random sampling 1000 different genome combinations of n genomes out of 67 genomes. Blue bars are the standard deviation of the medians. The red bars are weights used for model fitting and the red curve is the fitting result. b) Pan genome modeling of S. mutans genomes using three models, y = a + bx^c, y = a − bln(x + c) and y = a × e^− x/b + c (where a, b and c are parameters), represented by green, blue and red curves respectively. Black rectangles are the medians of the pan-genome sizes calculated by random sampling 1000 different genomes combination of n genomes out of 67 genomes, and black bars are the standard deviations of the medians.

Figure 4

Alignment of ComC and ComS amino acid sequences. a) Alignment of ComC amino acid sequences identified in S. mutans species using CLUSTALX. Conserved residues are marked with “*” above the figure. The diversity in the ComC sequences have been verified by PCR experiments (data not shown). b) BlastP alignment of the ComS sequence of S. mutans (identical among the eight S. mutans strains) with that of S. ratti DSM 20564 (No ComS was identified in S. sobrinus). “+” stands for similar amino acid residues.

Figure 5

Cluster structure of the mutacin-K8 production system across six S. mutans strains. The ORFs colored in yellow are the possible mutacin-K8 precursor genes. scnGEF: bacteriocin related ABC element; possible immunity system; scnK: histidine kinase of two component system; scnR: response regulator of two component system (Note: mutacin-K8 production system was failed to be identified in S. mutans NCTC 11060, S. mutans ATCC 25175, S. ratti DSM 20564 and S. sobrinus DSM 20742).

Figure 6

Central metabolism pathways of mutans streptococci. The orange lines represent enzyme reactions conserved across the mutans streptococci strains compared in this study, whereas the blue lines represent enzyme reactions specifically present (solid line) or absent (dashed line) in S. sobrinus DSM 20742. Reaction catalyzed by NAD⁺-specific malic enzyme (EC: 1.1.1.38) (green line) is absent in S. mutans NN2025 and S. mutans AC4446. Pyruvate-phosphate dikinase, catalyzing the interconversion of PEP and pyruvate (black line), is uniquely present in S. ratti DSM 20564.