Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of ?-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.
Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named T. cruzi I-VI. Although T. cruzi reproduces predominantly by binary division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the T. cruzi-specific 195-bp satellite DNA of TcI, TcII, TcIII, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that TcIII is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation.
Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of ?-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas, Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation.
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