Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group

Abstract

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.

FIG. 1.

Polymorphic nucleotide sites of ddlA alleles of S. salivarius and S. vestibularis: variable sites in each allele of the ddlA locus from the 27 S. salivarius strains and 9 S. vestibularis strains. The site numbers at the top are in vertical format, and S. vestibularis alleles are shaded.

FIG. 2.

Phylogenetic tree based on concatenated sequences of five housekeeping genes (ddlA, thrS, pyrE, dnaE, and sodA). The tree was constructed using the neighbor-joining method. Bootstrap values that are ≥80% are indicated at nodes. Scale bar = 0.005 nucleotide substitution per site. Disease-related isolates are indicated by an asterisk.

FIG. 3.

Split decomposition analysis of alleles present in 27 S. salivarius strains: split graphs for thrS, pyrE, and ddlA alleles. In some graphs several alleles are connected to each other by multiple pathways, forming an interconnected network and suggesting that there were recombination events. The numbers are allele numbers. Split graphs for ilvC, pepO, glcK, dnaE, and sodA alleles are shown in Fig. S2 in the supplemental material.

FIG. 4.

Phylogenetic relationships among 27 S. salivarius strains and 9 S. vestibularis strains. Trees are shown for glcK, ilvC, and pepO loci. Ssal, S. salivarius; Sves, S. vestibularis; p, pathogen; c, commensal; Spneumo, S. pneumoniae; Spsang, S. parasanguinis. The strain numbers correspond to ST numbers shown in Table 1. The trees were constructed by using the neighbor-joining method. Clusters and bootstrap values that are ≥80% are indicated. The scale bars indicate the number of nucleotide substitutions per site.

FIG. 5.

HGT events detected in S. salivarius pepO, ilvC, pyrE, and tkt genes. Regions where there are high and low levels of diversity are indicated by brackets, and the level of diversity is shown for each region. NS, no significant homology. (a) In the pepO region, 2,095-bp nucleotide sequences of LMG13109 cluster II and LMG14645 cluster I were compared. The regions were compared with the S. parasanguinis pepO region. (b) In the ilvC region, 2,765-bp nucleotide sequences of LMG13109 cluster II and LMG14645 cluster I were compared. The regions were compared with the S. pneumoniae ilvC region. (c) In allele 11 of the pyrE locus, a 234-bp DNA insertion containing 30 bp of S. salivarius IS1139 (gray box) and 204 bp of DNA whose function and origin are unknown is present and is flanked by a 157-bp duplication (arrows). (d) In the tkt region, 2,506-bp nucleotide sequences of S. salivarius LMG13109 and S. vestibularis LMG14645 were compared. orf1 exhibits the highest level of homology to str0310 from S. thermophilus.

FIG. 6.

Additive distance tree for tkt genes from streptococci and lactococci. Alignment was performed with ClustalW, and the tree was constructed by the flexible method. The levels of identity between the two clusters range from 54% to 58%, whereas the levels of identity are more than 73% for the genes within each cluster.