Confirmation of the presence of

Confirmation of the presence of H+-PPase genes in the genomes of the different protists tested was obtained by Southern blot analyses using the PCR-amplified DNA fragments as probes (Fig. 3). In the trypanosomatids L. major and T. cruzi and the euglenoid A. longa, a single band appeared in the filters (Figs. 3A and B); this was also the case for the rest of PCR-amplified DNA fragments that appear in Fig. 1 (results not shown). The use of two different probes both in P. tetraurelia and T. pyriformis showed a different hybridization pattern in every case (Fig. 3C), supporting the occurrence of two paralogous H+-PPase genes in these ciliates. Filters containing chromosomes of L. major and T. cruzi separated by CHEF electrophoresis were also hybridized with the appropriate probes. Only one signal appeared in the case of L. major, whereas a more complex pattern, with at least three hybridization bands, was obtained in T. cruzi (Fig. 4). This apparent disagreement with Southern blot results may be due to the extensive chromosome rearrangements and the aneuploidy reported to occur in T. cruzi. These features might generate homologous chromosomes of different sizes and, hence, the extreme karyotype polymorphism characteristic of this protozoan parasite [31]. Results shown in this paper demonstrate that genes encoding membrane-bound H+-PPase are present in the genome of a wide range of protozoa and suggest that, although in many cases they occur as a single gene, the presence of two different paralogous H+-PPase genes is relatively frequent in these microorganisms (cf. the cases of many plants and the apicomplexan P. falciparum[16]). It must be mentioned, however, that the experimental approach followed in this work may have some limitations, especially because primers were designed assuming that all H+-PPases contain the domains AGGI(A/S)EM and GGAWDNA. In any case, if there were other H+-PPase genes present in the organisms studied, they should be very different to those that were obtained, according to the analyses by Southern blots. In our view, this seems unlikely considering the experimental evidence available to date [6], [7], [16], [32]. Alignment of the amino Ketoconazole synthesis sequences deduced from the protozoan PCR-amplified DNA fragments obtained were analyzed and compared amongst them and also to other H+-PPase sequences found in public databases and genome projects. Several molecular phylogenetic methods were utilized, yielding qualitatively similar results. Fig. 5 shows a distance (neighbor-joining) phylogenetic tree which displays two major deeply rooted groups of catalytically different H+-PPase families with a broad distribution among prokaryotes, protists, and plants. These two H+-PPase groups are tentatively supported by biochemical differences found between the H+-PPases of two primitive bacteria that appear deeply rooted in the tree, namely, Thermotoga maritima (K+-stimulated) [6] and Chloroflexus aurantiacus (K+-insensitive) [J.R. Pérez-Castiñeira, M. Losada, A. Serrano, unpublished results]. The protist H+-PPase sequences reported in the present work clustered with the prototypical eukaryotic H+-stimulated K+-PPases of the green lineage, the algal and higher plant proteins [2], [6]. The only exception was the single H+-PPase found in the heterotrophic euglenoid A. longa, which clearly clustered with the bacterial K+-insensitive H+-PPases [4]. Although unexpected, this is not a surprising result since a second type of K+-insensitive H+-PPases, closely related to the homologous bacterial proteins (see Fig. 5), has been recently found in the plant A. thaliana and the apicomplexan P. falciparum[3], [16], [19]. Thus, although all the available evidence suggest that most protist H+-PPases constitute a compact protein cluster, consistent with the notion that these organisms are rather close in evolutionary terms, the possible existence of different evolutionary histories or lateral gene transfer between H+-PPases of different (micro)organisms cannot be ruled out. Finally, it should be noted that PPi-dependent proton pumping activities have been recently identified and biochemically characterized in the membranes of acidocalcisomes of Leishmania donovanii and T. cruzi and, consistent with our phylogenetic data, these proteins were shown to require potassium for full activity [8], [11]. It is also worthwhile pointing out that H+-PPases of trypanosomatids form a very compact monophyletic group that appears more closely related to the H+-PPase green lineage than any other protozoan H+-PPase (Fig. 5). This result is in agreement with recent reports on trypanosomes molecular evolution [33], [34], however, the full evolutionary implications of these results remain to be clarified.