Replacing and additive horizontal gene transfer in Streptococcus

Abstract

The prominent role of Horizontal Gene Transfer (HGT) in the evolution of bacteria is now well documented, but few studies have differentiated between evolutionary events that predominantly cause genes in one lineage to be replaced by homologs from another lineage ("replacing HGT") and events that result in the addition of substantial new genomic material ("additive HGT"). Here in, we make use of the distinct phylogenetic signatures of replacing and additive HGTs in a genome-wide study of the important human pathogen Streptococcus pyogenes (SPY) and its close relatives S. dysgalactiae subspecies equisimilis (SDE) and S. dysgalactiae subspecies dysgalactiae (SDD). Using recently developed statistical models and computational methods, we find evidence for abundant gene flow of both kinds within each of the SPY and SDE clades and of reduced levels of exchange between SPY and SDD. In addition, our analysis strongly supports a pronounced asymmetry in SPY-SDE gene flow, favoring the SPY-to-SDE direction. This finding is of particular interest in light of the recent increase in virulence of pathogenic SDE. We find much stronger evidence for SPY-SDE gene flow among replacing than among additive transfers, suggesting a primary influence from homologous recombination between co-occurring SPY and SDE cells in human hosts. Putative virulence genes are correlated with transfer events, but this correlation is found to be driven by additive, not replacing, HGTs. The genes affected by additive HGTs are enriched for functions having to do with transposition, recombination, and DNA integration, consistent with previous findings, whereas replacing HGTs seen to influence a more diverse set of genes. Additive transfers are also found to be associated with evidence of positive selection. These findings shed new light on the manner in which HGT has shaped pathogenic bacterial genomes.

Fig. 1.

Replacing and additive HGT. (A) Foreign DNA can be added to a recipient genome by a replacing HGT (left) or an additive HGT (right). (B) These types of transfers produce distinct phylogenetic signatures. In a replacing HGT, a segment of DNA is effectively overwritten by a homologous segment from another species, which causes a lineage in the phylogeny to be replaced by a transferred lineage. This type of transfer can be identified in gene-tree/species-tree reconciliation by the appearance of coinciding transfer and loss events (left tree). An additive HGT, on the other hand, leads to a transfer event that is not paired with a loss event (right tree). Of course, parallel losses can cause an additive HGT to appear similar to a replacing HGT. Therefore, in our analysis, we conservatively require that at least one descendant species (here, B) contains genes that both descend from (b₁) and do not descend from (b₂) a transferred gene when inferring an additive HGT event. It is worth noting that similar biological processes may contribute to both types of events—for example, an additive HGT may be associated with homologous recombination in flanking regions. Our interest here is in distinguishing between evolutionary processes that do (additive HGT) and do not (replacing HGT) tend to alter the size and gene composition of a genome.

Fig. 2.

Clonal frame inferred for the five genomes. This phylogeny was inferred using the ClonalFrame program (Didelot and Falush 2007). Branch lengths are in units of expected substitutions per site and are drawn to scale in the horizontal dimension. The labels for the ancestral nodes of the tree and the branches immediately ancestral to them (SPY, SDE, and SD) are used throughout the article. The outgroup species, SEE, was not part of the estimated clonal frame but was used in the parsimony-based analysis.

Fig. 3.

Heatmaps showing rates of replacing and additive transfers. Each cell of the heat map represents the base-2 logarithm of the ratio of the estimated number of recombination events to its prior expectation, for the corresponding donor (y axis) and recipient (x axis) branches. Cells in black indicate prohibited transfer events. (A) Replacing transfers, as inferred by the model-based approach. The plotted values represent average values across sampled recombinant graphs. The prior considers the clonal frame with branch lengths (fig. 2) and the global estimates of the three population parameters. Prohibited transfer events are ones for which the recipient branch is strictly older than the donor branch. An asterisk indicates statistical significance (P < 0.01). (B) Additive transfers, as inferred by the parsimony-based approach. The plotted log ratios reflect total numbers of inferred additive transfers across all gene families. The prior considers the clonal frame (with branch lengths) and the total number of events of each type inferred by the analysis. Prohibited transfer events are ones for which the recipient and donor branch do not share a common time interval. Significance levels are denoted by asterisks: one for P < 0.01, two for P < 0.005, and three for P < 0.001. See Materials and Methods for details.

Fig. 4.

Genome Browser tracks. Our inferences of replacing and additive horizontal gene transfers are summarized in new tracks in the Streptococcus Genome Browser (Suzuki et al. 2011). (A) The main browser display, with gene annotations for the selected reference genome (S. pyogenes strain MGAS315) shown in red and putative virulence genes highlighted in blue. The third track from top (in black) shows the putative non-recombining gene fragments analyzed by Mowgli. The next series of tracks shows the results of the ClonalOrigin analysis of replacing gene transfers. Each track in this series corresponds to a recipient lineage in the phylogeny (fig. 2) and describes the posterior probabilities along the genome of recombinant edges from all possible donor lineages (shown in different colors; see key). Here the putative virulence gene SpyM3_0465, a dipeptidase, shows strong evidence of a recombinant edge from the SPY to the SDE lineage, as well as some evidence of a SPY → SPY2 edge. The genome-wide multiple alignment obtained with Mauve is shown at bottom. (B) The gene tree and its reconciliation displayed after clicking on the gene fragment highlighted in orange. Note that the most parsimonious reconciliation of the tree estimated by RAxML involves a single SPY → SDE replacing HGT; however, this tree is also consistent with the combined influence of SPY → SDE and SPY → SPY2 replacing transfers, as inferred under the richer statistical model of ClonalOrigin.

Fig. 5.

Distribution of inferred gene duplication, loss, and transfer events across the six-species phylogeny. Numbers at nodes represent gene counts for extant species and estimated counts for ancestral species. Numbers on branches indicate inferred gene appearances (A*), duplications (D*), losses (L*), additive transfers (T*), and replacing transfers (R*). Transfer events are recorded on recipient branches (donors are not indicated). Singleton families (containing a single gene) are included as appearance events on external branches of the phylogeny.