Modelling the evolution of the archeal tryptophan synthase

Abstract

Background: Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate.

Results: In order to study the evolution of trp genes, completely sequenced archeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o) contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operon-based trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i) consisted of trpB2 genes located inside, the other (trpB2_a) of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium.

Conclusion: In archeal genomes, several stages of trpB evolution, TrpA/TrpB cooperation, and operon formation can be observed. Thus, archeal trp genes may serve as a model system for studying the evolution of protein-protein interactions and operon formation.

Figure 1

Phylogenetic tree of TrpB sequences. Using archeal and bacterial TrpB sequences, a multiple sequence alignment was generated and an unrooted phylogenetic tree was constructed. Proteins were labelled according to the naming scheme introduced in the Results section. Subtrees were marked according to the sequence type (TrpB1 or TrpB2). TrpB2 sequences span three subtrees; clustering is in agreement with the location of genes. TrpB2_o proteins are all encoded outside operons; 14 out of 16 originate from species that possess an operon-based trpB1 in addition. TrpB2_i proteins are encoded inside operons. Each of these genes is accompanied by a non operon-based trpB2. TrpB2_a sequences occur exclusively in genomes that have a single trpB2 gene or occur as a second trpB2 outside an operon in combination with a trpB2_i gene. The numbers are bootstrap values resulting from 1000 replications. Gene names are colour-coded. Blue colours indicate genes occurring in S2 (violet), s2 (light blue) and those i2_o2 species, which possess trpB2_a or trpB2_i genes (dark blue). Orange colours designate trpB2_i and trpB2_o genes. Red colours signify genes of i1_o2, S2, or s2 species, and green colours mark genes of s1 (light green) or S1 (dark green) species. The names of the two trpB1 copies occurring in N. pharaonis are printed in brown. For acronyms of species-types, see legend of Table 2. The length of the horizontal bar corresponds to 0.1 substitutions per site.

Figure 2

Phylogenetic tree of archeal and bacterial TrpA sequences. The two subtrees cluster genomes, which encode at least one trpB1 gene (S1, s1, i1_o1, or i1_o2 species) or which possess only genes of type trpB2 (S2, s2, i2_o2 species). The clusters were named TrpA1 or TrpA2, respectively. For abbreviations of sequence names, see Results. For colour code, see legend of Figure 1. For the acronyms of species-types, see legend of Table 2.

Figure 3

Phylogenetic tree of TrpD sequences. Archeal and bacterial protein sequences were used to construct the unrooted tree. The last letter of the acronyms indicates the taxonomical position of the species. "E" marks Euryarchaeota, "C" Crenarchaeota, and "B" bacterial species. The three TrpD sequences of N. pharaonis are designated as _1E, _2E, and _3E. For colour code and abbreviations, see legend of Figure 1.

Figure 4

Phylogenetic trees for TrpG and TrpE sequences. Archeal and bacterial protein sequences were used to construct the unrooted tree. The last letter of the acronyms indicates the taxonomical position of the species. "E" marks Euryarchaeota, "C" Crenarchaeota, and "B" bacterial species. The two TrpE sequences of N. pharaonis are designated as _1E, _2E. For colour code and abbreviations, see legend of Figure 1.

Figure 5

Multiple sequence alignment of TrpB sequences. Representatives of the four groups of TrpB sequences were aligned. Ssolfa_o2C, Ptorri_o2E, and Tacido_S2E represent TrpB2_a sequences. Ptorri_i2E, Ssolfa_i2C, and Stokod_i2C represent TrpB2_i, Maceti_o2E, Afulgi_o2E, and Tmarit_o2B represent TrpB2_o sequences. Maceti_i1E, Tmarit_i1B, Afulgi_i1E, and Pfurio_i1E represent TrpB1 sequences. The 2D-structural elements of Pfurio_i1E, as deduced from the PDB file 1WDW, are shown below the sequences, and residues involved in protein interaction with TrpA (I) are assigned under 'Interface'. The line Jpred (top) lists a 2D-prediction of Ssolfa_o2C generated by using the Jpred server [37]. Residues in bold face printing are conserved; black residues are strictly, grey residues are less strictly conserved. Active site residues are plotted in italics; residues in contact with ligands are underlined. These data were deduced from the PDBsum pages [56] and the PISA server [58] of the EMBL-EBI. Residues printed in boxes were predicted by SDPpred [38] as being specific for TrpB1 or TrpB2. See legend of Figure 1 for an explanation of sequence acronyms.

Figure 6

Multiple sequence alignment of TrpA sequences. Ssolfa_i2_o2C, Aperni_i2_o2C, and Ptorri_i2_o2C represent TrpA sequences from i2_o2 species possessing both an operon-based and a non operon-based trpB2. Tacido_s2E represents a species having exclusively an operon-based trpB2. Ecoli_s1B and Tmarit_i1_o2B represent bacterial TrpA proteins. Mstadt_s1E is from a species possessing exclusively an operon-based trpB1 gene, Mmazei_i1_o2E, Afulgi_i1_o2E and Pfurio_i1_o2E are TrpA sequences from i1_o2 species possessing an operon-based trpB1 and a non operon-based trpB2 gene. Presumably, these TrpA1 proteins interact with a protein of type TrpB1. Below the alignment, the 2D-structure of TrpA of P. furiosus (Pfurio_i1_o2E), and residues involved in protein interaction with its TrpB1 (I) are given. The line named Jpred lists a 2D-prediction of Ssolfa_i2_o2C generated by using the Jpred server [37]. Residues printed in bold are conserved; black residues are strictly, grey residues are less strictly conserved. Active site residues are plotted in italics. These data were deduced from the PDBsum pages [56] and the PISA server [58] of the EBI. Residues printed in boxes were predicted by SDPpred [38] as being specific for the two TrpA species.

Figure 7

Gene organisation of archeal and bacterial trp gene clusters. Each panel A – F represents the occurrence and orientation of trp genes in the genomes of organisms listed in the second column. The third column gives the species-type and the taxonomical lineage. "E" marks Euryarchaeota, "C" Crenarchaeota, and "B" bacteria. A vertical double line (Panels A and D) indicates borders of gene clusters separately located in the genome. Open arrows represent hypothetical genes. The arrows are not to scale; gaps of arbitrary length were inserted between genes to allow the alignment of arrows. For acronyms of species-types, see legend of Table 2.

Figure 8

A parsimonious reconstruction of predecessors. The phylogenetic tree is based on 16S rRNA sequence comparisons (after Fig. 2 of [49]). For all modern species, their species-type and evolutionary events leading most plausibly from the ancestral predecessor to the current genome content are added. The most probable species-type of predecessors for Crenarchaeota, Euryarchaeota, and Bacteria is given next to the grey circles. Abbreviations for events changing genomic content: Li1 or Lo2, (L)oss of the operon-based trpB1 or the non operon-based trpB2 gene, respectively. Ti1 (T)ranslocation of the operon based trpB1, Di1 (D)uplication of the operon-based trpB1. The colour of species names indicates the habitat: Hyperthermophiles are given in red, thermoacidophiles in orange, thermophiles in pink, mesophiles in green, halophiles in blue, and species living in a both hyperthermophilic and halophilic environment are given in purple.

Figure 9

Alternative models of trpB evolution. Model A assumes that a single and intermediate trpB* gene existed in the last universal common ancestor (LUCA) of bacteria and archaea. The evolution of the trpB2 gene is considered an archeal and that of the trpB1 gene is considered a bacterial invention. The occurrence of trpA1 and trpB1 genes in archaea and of trpB2 genes in bacteria are explained by a twofold horizontal gene transfer (HGT). A duplication of trpB2 in an ancient archeal genome has been postulated to explain the existence of the non operon-based trpB2. Models B and C propose two alternatives for the evolution of the LUCA. Model B assumes that the evolution trpB2 → trpB1 occurred in an early bacterial species after the divergence of bacteria and archaea. The replacement of linkage group trpB2A2 by trpB1A1 via HGT was postulated to account for the euryarcheal predecessor of type i1_o2. Model C assumes that the evolution trpB2 → trpB1 occurred before the divergence of bacteria and archaea. Hence, the replacement of an operon-based trpB1 by a trpB2 gene and the evolution trpA1 → trpA2 was postulated for the crenarcheal ancestor. For acronyms of species-types, see legend of Table 2. Distances are arbitrary and do not represent evolutionary time intervals. Stars indicate events of genomic rearrangements, circles filled in grey represent ancient predecessors.

Figure 10

Composite model of trpB evolution. Upon duplication and integration of an ancient trpB2 gene into the trp operon, the last universal common ancestor (LUCA) of bacteria and archaea was of species-type i2_o2. In a bacterial ancestor, the evolution of a linkage group trpB1A1 occurred. Via horizontal gene transfer (HGT), an euryarcheal ancestor acquired this linkage group, which gave rise to a predecessor of type i1_o2. Thermoplasmata acquired trpA2 and trpB2 genes in an ancient event of HGT. For all taxonomical orders, species-types of current species are given. S2 species possess exactly one, non operon-based trpB2 gene, s2: ditto, the gene is located inside the trp operon. trpB1 was treated analogously. i2_o2 are species possessing a trpB2 gene inside and a second trpB2 outside the operon, i1_o2 are species with an operon-based trpB1 and a non operon-based trpB2, and i1_o1 are species possessing an operon-based and at least one non operon-based trpB1.