Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi

Abstract

We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates' gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core-genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi-R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus.

Keywords: Actinobacteria; Corynebacteriales; Rhodococcus equi; comparative genomics; genome diversity and evolution; pangenome analysis; phylogenomics.

Fig. 1.—

Genomic similarity of Rhodococcus equi isolates. BLASTn alignment of 28 draft genomes (inner rings) (supplementary table S1, Supplementary Material online) against the complete 103S chromosome (Letek, et al. 2010). Outermost two rings, 103S genes in forward and reverse strands. E value cutoff = 0.1. The predominant red colour in the aligned sequences indicates BLAST hit ≥ 98% identity. Alignment gaps tend to coincide with regions of low G + C content in the 103S genome (innermost plot), many identified as HGT islands (arrowheads) by Alien_Hunter (Vernikos and Parkhill 2006). Drawn with CGViewer Comparison Tool (Grant et al. 2012).

Fig. 2.—

Rhodococcus equi core- and pangenome. (A) Pangenome distribution into strict core (present in 100% of isolates), soft-core (95% of isolates), cloud (≤2 genomes, cutoff defined as the class next to most populated noncore HGC) and shell (rest of HGCs). (B) Size estimation of core genome (left) and pangenome (right) by sequential sampling of n genomes in 10 random combinations using Tettelin exponential decay function fit (orthology threshold ≥50% for C and S) (Tettelin et al. 2005). Analyses in (A) and (B) performed with Get_Homologues (Contreras-Moreira and Vinuesa 2013). (C) Distribution of accessory genes in R. equi isolates. The (manually curated) complete 103S genome (Letek et al. 2010) was subjected to automated annotation as a control; the lower number of accessory genes in the manually annotated 103S sequence (n=667) suggests that the gene content is overestimated in the draft genome sequences. (D) KEGG categories of core and accessory genome HGCs. Only 15.6% of the accessory genes could be categorized versus 45.2% for the core genome, indicating that the accessory genome is a source of functional innovation in R. equi.

Fig. 3.—

Rhodococcus equi core–genome phylogeny. ML trees inferred using RealPhy (Bertels et al. 2014). Nodes indicate bootstrap support from 500 replicates. Scale bars indicate substitutions per site. (A) Unrooted tree with R. equi subclades (a–f) highlighted in different colours. (B) Unrooted tree as in (A) including the genome of the closely related species R. defluvii Ca11^T (GenBank assembly accession GCA_000738775.1) to illustrate the tight clustering of R. equi strains (see also supplementary fig. S6, Supplementary Material online). (C) Same tree as in (B) rooted with R. defluvii Ca11^T. Tips show strain name, source of isolation (host, geographic origin) and plasmid type (confirmed by sequence analysis: A, equine pVAPA; B, porcine pVAPB; N, ruminant pVAPN; –, no plasmid; a detailed comparative analysis of the virulence plasmid genomes will be reported elsewhere). Arrowheads indicate the reference genome strain 103S (Letek et al. 2010) and the type strain of R. equi (DSM 20307^T). Rhodococcus equi isolates are split into two major lineages, I and II.

Fig. 4.—

Whole-genome Corynebacteriales phylogeny. Constructed with PhyloPhlAn (Segata et al. 2013) using the genomes listed in supplementary table S3, Supplementary Material online. Streptomyces albus NBRC 1304^T was used as outgroup for tree rooting. Type strains are indicated by a T. All clades in the tree have been collapsed except the Rhodococcus equi–R. defluvii sublineage of Rhodococcus suclade 3. All nodes are strongly supported; see supplementary figure S7, Supplementary Material online, for a detailed tree with bootstrap values. Rhodococcus genus is in red, numbers designate major subclades (with letter suffix for sublineages). In blue, the genome of the type strain of R. rhodnii NRRL B-16535^T (GenBank assembly accession GCA_000720375.1) probably represents a case of strain mix-up or sequence mislabelling.