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RESEARCH LETTER A molecular probe for Basidiomycota : the spermidine synthase-saccharopine dehydrogenase chimeric gene Claudia G. León-Ramı́rez1, Laura Valdés-Santiago1, Eduardo Campos-Góngora1,2, Lucila Ortiz-Castellanos1, Elva T. Aréchiga-Carvajal3 & José Ruiz-Herrera1 1 Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico; 2Centro de Investigación en Nutrición y Salud Pública, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico; and 3Departamento de Microbiologı́a e Inmunologı́a, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico Correspondence: José Ruiz-Herrera, Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico. Tel.: 152 462 623 9652; fax: 152 462 624 5849; e-mail: [email protected] Received 7 January 2010; revised 1 June 2010; accepted 13 August 2010. Final version published online 15 September 2010. Abstract By means of an in silico analysis, we demonstrated that a previously described chimeric gene (Spe-Sdh) encoding spermidine synthase, a key enzyme involved in the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity of this procedure was tested using different fungal species. As expected, positive results were obtained only with Basidiomycota species, whereas no amplification was achieved with species belonging to other fungal phyla. DOI:10.1111/j.1574-6968.2010.02099.x MICROBIOLOGY LETTERS Editor: Jan Dijksterhuis Keywords Basidiomycota; spermidine synthase; saccharopine dehydrogenase; chimeric gene; molecular probe. Introduction Traditional available methods to identify and taxonomically describe fungal isolates are mainly based on morphological characteristics. In the specific case of Basidiomycota, the growth characteristics and/or pigmentation of the colonies in different media were used to distinguish some species (Dowson et al., 1988; Burgess et al., 1995). Other techniques involve the use of selective inhibitors or indicator substrates (Thorn et al., 1996). These methods have the disadvantages of being time-consuming and may lack accuracy. On the other hand, molecular methods have proved to be specific, sensitive, and rapid (Gardes & Bruns, 1996; Prewitt et al., 2008; Nicolotti et al., 2009). Amplification of ITS or Intergenic Spacer Regions of the rDNA sometimes combined with restriction analyses have been used to identify mycorrhizal, wood decay, and rust Basidiomycota species (Gardes & Bruns, 1993; Erland et al., 1994; Prewitt et al., 2008). FEMS Microbiol Lett 312 (2010) 77–83 Detection of specific genes has also been used as molecular markers, for example PCR analysis of genes encoding rRNA and intron determination in CHS genes (genes encoding chitin synthases) (Mehmann et al., 1994), or in Gpd, the gene encoding glyceraldehyde-3-phosphate dehydrogenase (Gardes et al., 1990; Mehmann et al., 1994; Kreuzinger et al., 1996). Although in general, these molecular methods may be useful in identifying some species, none of them cover most Basidiomycota, and may even render false positives. Previously, we reported the presence of a bifunctional gene encoding spermidine synthase (Spe) and saccharopine dehydrogenase (Sdh) in the Basidiomycota fungus Ustilago maydis, confirming previous data from Cryptococcus neoformans (Kingsbury et al., 2004). This gene contains a 5 0 -region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3 0 -region encoding Sdh (Valdés-Santiago et al., 2009). Apparently, this chimeric gene is specific to Basidiomycota, because in a preliminary 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 78 search, it could not be identified in several Ascomycota species. Spe catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Regarding lysine, it is known that fungi synthesize it via their exclusive mechanism, the a-aminoadipate pathway (see Xu et al., 2006). Sdh, also called saccharopine reductase, catalyzes the penultimate step in this pathway (Bhattacharjee, 1992). In the present work, we have performed an exhaustive analysis for the presence of a homologous gene in those Basidiomycota species whose genome has been sequenced, in other fungal taxa, and in the rest of living organisms reported in data banks. With the results obtained and the experimental data of gene amplification by PCR in different species, we propose the use of this gene as a molecular marker for Basidiomycota in general. Materials and methods Strains used and growth conditions Yarrowia lipolytica P01A was obtained from Claude Gaillardin (INRA), Saccharomyces cerevisiae S288C was obtained from American Type Culture Collection (ATCC 26108), Mucor rouxii IM80 (ATCC 24905) was obtained from Salomón Bartnicki-Garcia (University of California, Riverside), Rhizopus oryzae 2672 was obtained from CECT (Colección Española de Cultivos Tipo), U. maydis FB2 was obtained from Flora Banuett (California State University, Long Beach), Coprinus cinereus UAMH4103 was obtained from University of Alberta Microfungus Collection and Herbarium, Ustilago hordei 8A was obtained from ATCC (90511); Ganoderma lucidum, Ganoderma sp., Schizophyllum commune, Pleurotus ostreatus, Rhizoctonia solani, Agaricus bisporus, Ustilago cynodontis, Tilletia foetida, and Bjerkandera adusta belong to the collection from Laboratorio de Micologı́a (Universidad Autónoma de Nuevo León, Monterrey, NL, Mexico). Fungal strains were maintained in 50% glycerol at 70 1C. For propagation, strains were inoculated in liquid YPG medium [yeast extract (Difco), 2%; peptone (Difco), 1%, and glucose (Merck), 1%] and incubated at 28 1C for 18 h under shaking conditions (150 r.p.m.). Escherichia coli strain ElectroMAXTMDH10BTM (Invitrogen Life Technologies) was used for transformation with the PCR-amplified products cloned previously in TOPOTM4 vector (Invitrogen). It was grown in Luria–Bertani (LB) liquid medium (Becton Dickinson) at 37 1C or on plates of the same medium containing 2% agar plus the antibiotics required for plasmid selection. C.G. León-Ramı́rez et al. sterile distilled water. DNA was isolated following the glass beads lysis protocol as described by Hoffman & Wriston (1987). In silico search of homologs of the chimeric gene and their alignment Searches for homologs were performed using as query the whole sequence of the U. maydis chimeric gene (EMBL accession number FN178523; Valdés-Santiago et al., 2009) at the NCBI (http://www.ncbi.nlm.nih.gov), and The Joint Genome Institute, U.S. Department of Energy (http:// genome.jgi-psf.org), using BLASTN, BLASTP or TBLASTN programs (Altschul et al., 1990). Sequence alignments of putative chimeric Spe-Sdh gene fragments were performed using CLUSTAL W (Thompson et al., 1994). Degenerate primers design Degenerate primers were designed at the most highly conserved regions to the chimeric genes based on their alignment. The sequences of these primers are the following: forward primer (F-CHIM) 5 0 -CA(G/A)GA(G/A)ATGAT(C/T)GC (T/C/G) CA (T/C)(C/T)T(C/T/G/A)CC-3 0 , and reverse primer (R-CHIM) 50 -(C/T)T(C/T/G/A)GG(C/G/A)T(A/C)(C/A)AA (G/A)TTCTC (G/C/A/T)TGGTC(G/C/T/A)(T/C/G)C-3 0 . PCR conditions A standard protocol was performed for PCR amplification: an initial denaturation at 94 1C for 5 min, followed by 35 cycles with the following program: DNA denaturation at 94 1C for 1 min; primer annealing at 60 1C for 1 min, DNA extension at 72 1C for 2 min, with a final extension cycle at 72 1C for 10 min. The PCR reaction products were separated by electrophoresis in 0.7% agarose gels and stained with ethidium bromide. Cloning and sequencing of PCR products PCR products amplified with the degenerate primers described above were either cloned or not cloned into a TOPOTM4 vector (Invitrogen) and sequenced using an ABI PRISM Model 370 sequencer (Applied Biosystems). Cloned fragments were sequenced from the vector with the universal primers, whereas not cloned fragments were sequenced using the degenerate primers. Results and discussion DNA isolation Identification of homologs of the chimeric gene Cells were grown as described above for 18 h, harvested by centrifugation, and washed twice by centrifugation with During the search of homologs as described in Methods, we observed that only fungi belonging to Basidiomycota 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c FEMS Microbiol Lett 312 (2010) 77–83 79 Chimeric gene in Basidiomycota contained homologs of the Spd-Sdh chimeric gene. No homologs were detected, not only in the rest of the fungal groups but also in any other living organism. Among the sequences identified, besides the U. maydis gene described previously (Valdés-Santiago et al., 2009), the ones that possessed NCBI accession numbers corresponded to the following species: Coprinus cinereus (EAU83678), Malassezia globosa (EDP44132), Laccaria bicolor (EDR1322), and Cryptococcus neoformans (EAL19736). Other species possess homolog genes encoding proteins with the identification number (protein ID) from the Joint Genome Institute: Heterobasidion annosum 7(34012), Tremella mesenterica (74272), Sporobolomyces roseus (23418), Pleurotus ostreatus (172366), Postia placenta (107976), Melampsora laricis populina (73077), and Phanerochaete chrysosporium (9263). The Puccinia graminis homolog corresponded to PGTG_06954 (Broad Institute). Alignment of chimeric genes with their corresponding independent homologs A comparison of the chimeric gene Spe-Sdh nucleotide sequence from the Basidiomycota species reported in databases with the independent Spe and Sdh genes present in species of Ascomycota (S. cerevisiae and Aspergillus fumigatus) revealed the presence of two distinct regions. The one located at the 5 0 - region showed high homology with the Spe genes, whereas the one present at the 3 0 -region was homologous to the Sdh genes; both were linked through a region of approximately 60 nucleotides without homology (not shown). As expected, the alignment of amino acid sequences encoded by these genes showed the same pattern of homology, demonstrating the high preservation of the gene in the Basidiomycota (not shown). With these data we designed degenerate primers to be used for PCR amplification of the chimeric genes. The forward primer was selected (a) 5′ 3′ TTGGGCTACAAGTTCTCTTGGTCGTC CTTGGATCAAAGTTCTCGTGGTCGTC CTCGGCTACAAGTTCTCATGGTCGGC CTCGGCTACAAGTTCTCGTGGTCGTC CTCGGCTACAAATTCTCCTGGTCCTC CTCGGCTACAAGTTCTCCTGGTCGTC CTCGGGTACAAGTTCTCTTGGTCCCC CTTGGCTACAAGTTCTCGTGGTCTTC CTGGGCTACAAATTCTCGTGGTCTTC TTAGGATACAAGTTCTCCTGGTCATC * ** ** ** ***** ***** * 1 CAAGAGATGATCGCTCACTTGCC 2 CAAGAGATGATCGCTCACCTTCC 3 CAGGAGATGATCGCTCATCTACC 4 CAGGAAATGATTGCACACCTCCC 5 CAAGAGATGATCGCCCATCTCCC 6 6CAGGAGATGATTGCTCACCTTCC 7 CAGGAGATGATCACTCATCTTCC 8 CAGGAGATGATCGCCCATCTCCC 9 CAAGAGATGATCGCTCATTTGCC 10 CAGGAAATGATTGCGCATATACC ** ** ***** ** ** * ** (b) Kb M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M 2 1.6 1 Fig. 1. Amplification of a fragment from the Spe-Sdh chimeric gene by PCR. (a) Schematic representation of the multiple alignment of putative Spe-Sdh chimeric genes reported in the GenBank. Black box, Spe region of the gene; speckled gray box, inter ORF region; white box, Sdh region of the gene. Location of the sequences selected for degenerate primer design are schematically indicated. 1, Coprinus cinereus; 2, Sporobolomyces roseus; 3, Postia placenta; 4, Malassezia globosa; 5, Laccaria bicolor; 6, Ustilago maydis; 7, Cryptococcus neoformans; 8, Phanerochaete chrysosporium; 9, Puccinia graminis; 10, Melampsora laricis populina. (b) Gel electrophoresis of PCR products amplified with the degenerate primers from DNA of different species. M, molecular marker; lane 1, Ustilago maydis; lane 2, Bjerkandera adusta; lane 3, Tilletia foetida; lane 4, Ustilago cynodontis; lane 5, Ustilago hordei; lane 6, Rhizoctonia solani; lane 7, Schizophyllum commune; lane 8, C. cinereus; lane 9, Pleurotus ostreatus; lane 10, Ganoderma lucidum; lane 11, Ganoderma sp.; lane 12, Agaricus bisporus; lane 13, Saccharomyces cerevisiae; lane 14, Yarrowia lipolytica; lane 15, Rhizopus oryzae; and lane 16, Mucor rouxii. FEMS Microbiol Lett 312 (2010) 77–83 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c C.G. León-Ramı́rez et al. Fig. 2. Alignment of the sequences encoded by the fragments of the chimeric genes belonging to different Basidiomycota species amplified in this work. 80 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c FEMS Microbiol Lett 312 (2010) 77–83 81 Chimeric gene in Basidiomycota at the 3 0 -end of the region with homology to Spe, and the reverse primer was designed from the homologous region at the 5 0 -end of the Sdh, in such a way that the amplification fragment covered the nonhomologous region that separates both coding regions (see Fig. 1a). Specificity of degenerate primers, and cloning and sequencing of the PCR products The sequences of the fragments were deposited in GenBank, with the following accession numbers: Ustilago cynodontis, FN646089; Tilletia foetida, FN646090; Bjerkandera adusta, FN646091; Rhizoctonia solani, FN822770; Schizophyllum commune, FN822771; Ustilago hordei, FN822772; Ustilago maydis, FN822773; Coprinus cinerea, FN822774; Pleurotus ostreatus, FN822775; Ganoderma lucidum, FN822776; Agaricus bisporus, FN827330; and Ganoderma sp., FN827329. Phylogenetic analyses Using the PCR conditions described above and the designed degenerate primers, it was possible to amplify DNA fragments of the predicted size from genomic DNA of all the Basidiomycota species tested (see Materials and methods), whose genomes have been sequenced or not, that represented the three subphyla from Basidiomycota. The size of the fragments (around 1300 bp) coincided with the expected values. On the other hand, and as expected, no such amplification occurred when DNA from Ascomycota or Zygomycota species was used as template (Fig. 1b). The PCR products corresponding to the Basidiomycota species analyzed in this work were sequenced. Alignment of the encoded sequences revealed their high conservation (Fig. 2). Additionally, the encoded sequences of the amplified fragments from Basidiomycota species whose genomes had been previously sequenced were compared with those existing in their corresponding data banks. The results obtained confirmed the fidelity of the PCR amplification (Table 1). The differences observed can be explained by the fact that different isolates were used in these studies. The sequences of the regions corresponding to the fragments amplified by PCR from the Spe-Sdh genes obtained in this Table 1. Comparison of chimeric Spe-Sdh sequences determined in this work with corresponding sequences reported in GenBank Species (ID) Identity Ustilago maydis (http://www.ncbi.nlm.nih.gov/protein/71023471)w Coprinus cinereus (XP_001838101.1)w Pleurotus ostreatus (ID 114149)z Agaricus bisporus (ID 188516)z Schizophyllum commune (ID 257519)z 97% (357/367) 96% (356/369) 96% (397/410) 85% (308/360) 99% (407/409) Percentage of amino acid identity (no. of identical residues/no. of residues compared). Sequences obtained from NCBI. z The Joint Genome Institute. w Puccinomycotina Ustilaginomycotina Fig. 3. Dendrogram of the sequences corresponding to the amplified fragments of the putative Spe-Sdh genes from species representative of the three Basidiomycota subphyla. Sequences of the fragments from Ustilago cynodontis, Tilletia foetida, Bjerkandera adusta, Agaricus bisporus, Rhizoctonia solani, Schizophyllum commune, Ustilago hordei, Ustilago maydis, Coprinus cinerea, Pleurotus ostreatus, Ganoderma lucidum and Ganoderma sp. were obtained in this work; the rest were obtained from databases (see Materials and methods). FEMS Microbiol Lett 312 (2010) 77–83 Agaricomycotina 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c c 58 Malassezia 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Ganoderma sp. bisporus Agaricus adusta Bjerkandera lucidum Ganoderma ostreatus Pleurotus 56 47 62 49 60 77 61 81 69 77 67 Coprinus cinereus 58 57 57 54 77 63 63 64 62 81 64 62 81 79 58 Ustilago maydis 56 Ustilago hordei commune Schizophyllum 62 60 solani 62 Rhizoctonia 62 54 62 68 Tilletia foetida cynodontis Ustilago mesenterica Tremmella annosum Heterobasidion larici-populina Melampsora graminis Puccinia 65 62 Phanerochaete chrysosporium 62 Laccaria bicolor globosa 64 Postia placenta roseus 100 55 46 57 49 54 51 69 67 56 72 73 72 51 57 57 56 56 57 100 placenta globosa roseus Sporobolomyces 100 chaete Phanero- 73 66 78 65 79 74 57 57 80 62 62 63 59 82 62 60 77 100 bicolor 72 61 86 64 75 67 59 59 74 64 64 65 60 77 65 63 100 sporium Malassezia Laccaria chryso- Postia myces Sporobolo- 57 48 61 50 57 56 54 53 61 58 59 58 51 62 80 100 59 51 64 51 63 60 56 53 62 59 60 60 54 64 100 graminis populina Puccinia sora larici- MelampTremmella Ustilago Tilletia 74 66 78 66 78 71 61 60 79 65 66 66 61 100 58 48 61 51 59 52 52 53 61 58 57 57 100 60 49 65 52 60 53 91 94 63 97 95 100 60 48 64 52 60 53 96 91 62 94 100 annosum mesenterica cynodontis foetida basidion Hetero- Table 2. Homology matrix of the Spe-Sdh gene fragments from different Basidiomycota species 60 48 65 52 59 52 90 97 62 100 solani ctonia Rhizo- 72 63 76 64 75 70 58 58 100 56 46 59 50 54 50 89 100 56 45 58 51 54 50 100 maydis Ustilago Ustilago commune hordei phyllum Schizo- 66 64 68 60 71 100 cinereus Coprinus erma andera Ganod- Bjerk- 70 62 78 62 100 84 56 65 100 73 61 100 ostreatusa lucidum adusta Pleurotus Gano- 61 100 100 bisporus sp. Agaricus derma 82 C.G. León-Ramı́rez et al. FEMS Microbiol Lett 312 (2010) 77–83 83 Chimeric gene in Basidiomycota study, and those reported in the databases, were used for the construction of a phylogenetic tree. The results obtained showed the phylogenetic relationship (Fig. 3) and similarity (Table 2) among the fragments belonging to the different species analyzed that represent the three subphyla from Basidiomycota: Puccinomycotina, Ustilaginomycotina, and Agaricomycotina, respectively. It may be also observed that the dendrogram obtained (Fig. 3) coincides with the phylogenetic division of the Basidiomycota subphyla, confirming the unique and common origin of the chimeric gene in this phylum. It is interesting to recall that the chimeric gene encoding Spe and Sdh is specific to Basidiomycota, whereas biosynthetic Sdh genes from other non-Basidiomycota fungal species exist in a free independent form. Additionally, the catabolic Sdh gene may be chimeric with the gene encoding lysine ketoglutarate reductase, which is the next enzyme involved in the catabolism of lysine. In other organisms, the catabolic Sdh gene may be bound to a motif that is related to alanine dehydrogenase. The reasons behind the appearance of the Spe-Sdh chimeric gene are obscure, because there does not appear to be a direct relationship between the metabolism of polyamines and lysine. The event should have occurred in a common ancestor of Basidiomycota, as it is present in all the modern members of the phylum, and as hypothesized previously (Valdés-Santiago et al., 2009), it is possible that both genes remained associated throughout evolution, because the high cost of losing simultaneously the pathways leading to the synthesis of different essential metabolites. The results presented here indicate that, as mentioned repeatedly, the Spe-Sdh chimeric gene is specific to Basidiomycota, being absent not only in any other fungal group but also in any other eukaryotic taxa. Therefore, it is a specific marker of the phylum Basidiomycota, and its detection undoubtedly will be the most useful method for the validation of any isolate belonging to this phylum. Acknowledgements The present work was partially supported by Consejo Nacional de Ciencia y Tecnologı́a (CONACYT), Mexico. L.V.S. is a doctoral student supported by a fellowship from CONACYT. 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