Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements

Abstract

Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.

FIG. 1.

Phylogeny of DAHP I and DAHP II genes. (A) Phylogeny of DAHP I gene, calculated from an amino acid alignment of 45 sequences and 327 characters. The node demonstrating oomycete and fungal monophyly is shown with a box. (B) Phylogeny of DAHP II, calculated from an amino acid alignment of 35 sequences and 372 characters. The node demonstrating the monophyly of arom possessing taxa and the putative HGT with S. usitatus is shown with a box; the node showing separation of the land plants/diatom from the other eukaryotes is circled. This node also represents the best support for a putative HGT between Plantae and a proteobacterium. The topologies shown are the results of the Bayesian topology search arbitrarily rooted on a eubacterial branch. Topology support values for this and all subsequent phylogeny figures are illustrated when both bootstrap results were above 49% and are shown in the order Bayesian posterior probability/% 1,000 ML distance bootstraps/% 100 fast ML-PHYML bootstraps. A selection of eubacteria and the eukaryotes are labeled according to higher taxonomic classification (prokaryote taxa using line bars and eukaryotes using box bars). In addition, the eukaryotes are named in bold. These labeling conventions are also used in all subsequent figures.

FIG. 2.

Phylogeny of AroB. This phylogeny was calculated from an amino acid alignment of 87 sequences and 209 characters. The node showing separation of the Plantae away from the arom-possessing eukaryotes, indicative of an HGT from the β/γ-proteobacteria is circled. The node supporting monophyly of arom-possessing taxa, with the exception of T. thermophila and T. gondii, and demonstrating arom reduction in R. oryzae is boxed. The figure is illustrated using the same conventions as Fig. 1, with the addition that fusion genes are noted using square brackets and the shikimate gene fusion order is illustrated using the Aro convention. This convention for fusion genes will be used in all subsequent relevant figures.

FIG. 3.

Phylogeny of DHQase II, which encodes step 3 of the shikimate pathway. Type I 3-dehydroquinate dehydratase could not be reliably aligned and analyzed so is not included here. Phylogeny of DHQase II gene was calculated from an amino acid alignment of 54 sequences and 139 characters. The figure is illustrated using the same conventions as Fig. 1.

FIG. 4.

Phylogeny of AroE. (A) Phylogeny of AroE was calculated from an amino acid alignment of 97 sequences and 163 characters. The node demonstrating monophyly of the arom-possessing taxa and suggesting the reduction of arom in T. pseudonana is boxed. Note also the PheA domain (chorismate mutase EC 5.4.99.5) is fused to the AroE in Clostridium acetobutylicum. The node supporting the close relationship of the R. baltica and land plants is circled. Note that the presence of C. merolae as the immediate out-group to this clade prompted the analysis in panel B. (B) Phylogeny of the AroDE fusion gene calculated from an amino acid alignment of 16 sequences and 419 characters. In this analysis, 1,000 protein parsimony bootstrap analyses were also conducted to provide more data in view of the uncertainties of where C. merolae branches. The fourth support value is the result of the 1,000 protein parsimony bootstraps. Branching support within the land plants and the Chlamydiales is not shown, to reduce figure complexity. Alternative C. merolae branching position and bootstrap support is shown using the gray hatched line.

FIG. 5.

Phylogeny of AroK/L. The AroK/L phylogeny was calculated from an amino acid alignment of 88 sequences and 123 characters. The node supporting monophyly of arom-possessing taxa is boxed. The node showing separation of the Plantae away from the remaining eukaryotes and grouping with the cyanobacteria is circled. Note also that the HTH_XRE domain (Helix-turn-helix motif pfam01381) is fused to AroK in some proteobacteria.

FIG. 6.

Phylogeny of AroA. The AroA phylogeny was calculated from an amino acid alignment of 90 sequences and 259 characters. The node supporting monophyly of arom-possessing taxa is boxed. The node showing separation of the Plantae away from the arom-possessing eukaryotes and indicating an HGT from the β/γ-proteobacteria to the Plantae is circled.

FIG. 7.

Phylogeny of AroC. The AroC phylogeny was calculated from an amino acid alignment of 84 sequences and 267 characters. The node showing monophyly of arom-possessing taxa, despite this gene not being part of arom, is illustrated with a box. The node showing separation of the Plantae away from the remaining eukaryotes and grouping with the cyanobacteria is circled.

FIG. 8.

(A) Schematic of evolution of the eukaryotes and their shikimate pathway genes using the bikont/unikont eukaryote root (37, 48, 49). Shikimate pathway genes are colored and are in the gene fusion order as indicated in panel B. Mitochondria and nuclear organelles are marked M and N, respectively. Apicoplastid organelles are illustrated as a red capsule, plastids are colored green, and plastids of secondary endosymbiotic origin are colored green with a red border. Secondary endosymbiotic events, hypothetical loss events, and horizontal gene transfers are labeled and are given an arrow to infer the direction and target of the gene transfer. Note that the shikimate pathway genes are localized in the cell only to reflect the likely associated origin, i.e., not to demonstrate genome localization or functional localization. For example, the arom pathway probably arose at a similar time as the eukaryotic cytoplasm, so it is drawn in the cytoplasm; the Plantae shikimate pathway is likely to have arisen at a similar time to the plastid endosymbiosis, so it is drawn associated with the plastid, although in several cases origin is not directly from the plastid progenitor genome. (B) Key for the color coding of the shikimate pathway genes and illustration of the alternative naming for each gene/enzyme. A brief outline of the metabolic pathway is given.