The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Critical analysis of the topology and rooting of the parabasalian 16S rRNA tree

The morphological classification of the protozoan phylum Parabasala is not in absolute agreement with the 16S rRNA phylogeny. However, there are strong indications that tree-construction artifacts play a considerable role in the shaping of the 16S rRNA tree. We have performed rigorous analyses designed to minimize such artifacts using the slow-fast and taxa-exclusion methods. The analyses, which included new sequences from the genera Monocercomonas and Hexamastix, in most respects confirmed the previously suggested tree topology and polyphyly of Hypermastigida and Monocercomonadidae but detected one artificial cluster of long branches (Trichonymphidae, Pseudotrichonymphidae, Hexamastix, and Tricercomitus). They also indicated that the rooting of the phylum on the trichonymphid branch is probably wrong and that reliable rooting on the basis of current data is likely impossible. We discuss the tree topology in the view of anagenesis of cytoskeletal and motility organelles and suggest that a robust taxonomic revision requires extensive analysis of other gene sequences.     

Root of the Eukaryota tree as inferred from combined maximum likelihood analyses of multiple molecular sequence data

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.     

Phylogenetic Relationships of the Glycolytic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, from Parabasalid Flagellates

Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala. With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species. All sequences were colinear with T. vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH. All residues known to be highly conserved in this enzyme were present. The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods. The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages. T. vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp. and Tritrichomonas foetus formed the other. These data agreed with and/or were close to published reconstructions based on other macromolecules. They did not support the ancestral position of Monocercomonas sp. proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum. The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages. The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes. The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far. Received: 28 April 1997 / Accepted: 14 October 1997     

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.