1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways

The methionine salvage pathway.

<p>The enzyme names are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>, and compound names are from KEGG <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>. The reactions in black are known in bacteria <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>. The yeast pathway is indicated by blue gene names under the corresponding enzymes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Pirkov1" target="_blank">[9]</a>. Dashed lines indicate variants of the pathway (see text). In <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Ashida1" target="_blank">[5]</a> it was noted that the genes coding for mtnB and mtnC appear to be fused in <i>Arabidopsis thaliana</i>, and the genes for mtnB and mtnD appear to be fused in <i>Tetrahymena thermophila</i>, which indicates that the pathway in these organisms proceeds through the green and red reaction lines, respectively. We identified another fusion gene, between mtnK and mtnA, in <i>Tetrahymena</i> (red line).</p

Screenshot of a tblastn search of the mtnAK enzyme from <i>Tetrahymena</i> (XP_001031773) against the EST sequences from <i>Tetrahymena</i> in GenBank.

<p>The EST sequences TT1BI24TH (acc: FF565362; evalue = 1×10⁻¹²⁵) and TT1BI24TV (acc: FF565363; evalue = 2×10⁻¹²⁵) correspond to the 5′- and 3′-end of a single cDNA clone, indicating that the fusion protein is expressed in <i>Tetrahymena</i>.</p

Complementation experiment of yeast single knockout strains with mtnBD fusion gene from <i>Tetrahymena</i>.

<p>Cells were grown to late exponential phase in −Leu +Met liquid media, transferred to −Leu−Met for an overnight to deplete internal Methionine pool, and serial dilutions for each strain were prepared in a 96-well plate with 1×10⁸ cells/ml, 1×10⁷ cells/ml, 1×10⁶ cells/ml, 1×10⁵, 1×10⁴ cells/ml, and 1×10³ cells/ml. 3 µl of each diluted culture was spotted with a 96-well pin replicator onto (A) −Met−MTA (negative control plate). (B) +Met plates, (positive control). (C) −Met +MTA (5 mM) (experimental plate). The strains assayed are: 1. <i>mtnB</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 2. <i>mtnB</i>Δ + pGREG505; 3. <i>mtnC</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 4. <i>mtnC</i>Δ + pGREG505; 5. <i>mtnD</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 6. <i>mtnD</i>Δ + pGREG505; columns 7–12 are replicates of columns 1 through 6. After four days, none of the six yeast strains grew on the negative control plate. All six strains grew on the positive control plate. Only the strains transformed with pGREG/<i>SYN-MTNBD</i> (columns 1, 3, 5, 7, 9, 11) grew in the experimental plate.</p

Homologs of <i>B. subtillis</i> and yeast methionine salvage pathway enzymes in <i>Tetrahymena</i>.

<p>*Enzyme names and EC numbers are from KEGG (Kyoto Encyclopedia of Genes and Genomes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>.</p

Morphology, reproduction and karyology in the rare Andean Poa gymnantha

We report agamic reproduction in Poa gymnantha, an endemic species from the high Andes of Peru, Bolivia and Chile, known only from pistillate plants. This is the first description of apomixis in Poa from South America. The species is a decaploid (2n = 10x = 70) with a basic karyotype formula of 4 m + 3 sm chromosomes. Diagnostic characters, geographic distribution and taxonomic circumscription of the species are given, along with illustrations. The paper contributes to a better understanding of floral morphology and reproduction in this agamic taxon.Fil: Negritto, María de Los Angeles. Universidad de Concepción; ChileFil: Romanutti, Alejandra Aida. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Acosta, María Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Moscone, Eduardo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Cocucci, Alfredo Elio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Anton, Ana Maria Ramona. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin