Phylogenetic relationships of Malassezia species based on multilocus sequence analysis

Members of the genus Malassezia are lipophilic basidiomycetous yeasts, which are part of the normal cutaneous microbiota of humans and other warm-blooded animals. Currently, this genus consists of 14 species that have been characterized by phenetic and molecular methods. Although several molecular methods have been used to identify and/or differentiate Malassezia species, the sequencing of the rRNA genes and the chitin synthase-2 gene (CHS2) are the most widely employed. There is little information about the beta-tubulin gene in the genus Malassezia, a gene has been used for the analysis of complex species groups. The aim of the present study was to sequence a fragment of the beta-tubulin gene of Malassezia species and analyze their phylogenetic relationship using a multilocus sequence approach based on two rRNA genes (ITS including 5.8S rRNA and D1/D2 region of 26S rRNA) together with two protein encoding genes (CHS2 and beta-tubulin). The phylogenetic study of the partial beta-tubulin gene sequences indicated that this molecular marker can be used to assess diversity and identify new species. The multilocus sequence analysis of the four loci provides robust support to delineate species at the terminal nodes and could help to estimate divergence times for the origin and diversification of Malassezia species.

Diversidade de populações de Phyllosticta spp. de goiabeiras e de mangueiras em diferentes ambientes

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Uso de probiótico e óleos essenciais na ração sobre a microbiota intestinal, atividade de enzimas digestivas e a expressão de genes relacionados aos processos de digestão e absorção de nutrientes em frangos

Pós-graduação em Zootecnia - FCAV

Pollination systems in Pogonieae (Orchidaceae: Vanilloideae): A hypothesis of evolution among reward and rewardless flowers

The tribe Pogonieae of Vanilloideae (Orchidaceae) consists of six genera, including Pogoniopsis, a mycoheterotrophic taxon with morphological characteristics distinct from the remaining of the tribe. A hypothesis about the phylogeny of the tribe was inferred, involving all currently recognized genera, based on isolated and combined sequence data of 5.8S, 18S and 26S (nrDNA) regions using parsimony and Bayesian analyses. Phylogenetic analyses show that inclusion of Pogoniopsis turns the tribe Pogonieae paraphyletic. All analyses reveal that Pogoniopsis is closely related to members of Epidendroideae. The pantropical Vanilla is monophyletic if Dictyophyllaria is assumed as synonym of Vanilla. Members of Pogonieae are pollinated by several groups of solitary and social bees, two pollination systems being recognized: reward-producing and deceptive. The molecular phylogeny suggests that ancestrals related to Pogonieae gave rise to two evolutionary lines: a tropical one with reward production of flowers, and a predominantly temperate regions invading line with deceptive flowers. Reward-producing flowers characterize the South and Central American clade (=Cleistes), while deceptive pollination is prominent in the clade that includes North American-Asiatic taxa plus the Amazonian genus Duckeella. (C) 2012 Elsevier GmbH. All rights reserved.

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceara, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceara, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1 b and 1 c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1 -5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceara have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

Nicoletella semolina gen. nov., sp. nov., a new member of Pasteurellaceae isolated from horses with airway disease

Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape. DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N. semolina as a new genus in the family Pasteurellaceae. In all the phylogenetic trees constructed, N. semolina is on a distinct branch displaying approximately 5% 16S rDNA, approximately 16% rpoB, and approximately 20% infB sequence divergence from its nearest relative within the family Pasteurellaceae. High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N. semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous. The type strain of N. semolina is CCUG43639(T) (DSM16380(T)).

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.

Determinación de la contaminación con Zygosaccharomyces rouxii durante la elaboración de jugos de uva concentrados

El jugo de uva concentrado (JUC) es un commodity y por su carácter natural se utiliza para elaborar jugos mezclas, golosinas, dulces, mermeladas, jaleas, galletitas, pan, como edulcorante de bebidas gaseosas, y también en la industria farmacéutica. La producción de JUC constituye una parte importante de la industria vitivinícola argentina, siendo nuestro país el mayor exportador mundial de JUC durante el año 2014. El comercio internacional del JUC forma parte de mercados con una demanda que crece en forma sostenida. La industrialización de la uva para la obtención de jugos concentrados presenta varias etapas de procesamiento que incluyen tratamientos térmicos que afectan la microbiota presente en la materia prima. A pesar de esto, los productos no están exentos de presentar problemas microbiológicos que deterioran la calidad del mismo. El JUC es un alimento de humedad intermedia (aw 0,7-0,8), con elevada concentración de azúcares y bajo pH. La alteración de estos sustratos es causada por levaduras osmófilas, dentro de este grupo el género que se aísla con mayor frecuencia es Zygosaccharomyces sp. El objetivo del presente trabajo fue la identificación de puntos críticos de contaminación con levaduras osmófilas en plantas elaboradoras, identificando las especies presentes en los jugos de uva y las superficies asociadas a su concentrado. El conocimiento de los puntos críticos de contaminación permitiría la aplicación de medidas preventivas para aumentar la estabilidad microbiana de los JUC. Para ello se eligieron tres plantas concentradoras de jugo de uva y se muestrearon los jugos de uva pre-concentrados y concentrados y las superficies asociadas a su elaboración. Se realizó el recuento de levaduras osmófilas en el medio MY50G y la posterior identificación molecular de las levaduras presentes en todas las muestras mediante secuenciación del fragmento amplificado ITS1-5.8S-ITS2. Los resultados mostraron que Z. rouxii fue la especie encontrada en todas las muestras de jugo de uva pre-concentrado y concentrado y en la mayoría de los casos representó el 100% de las levaduras aisladas. También se evidenció que los períodos de almacenamiento del jugo de uva pre-concentrado y concentrado fueron claves para que la población de Z. rouxii aumentara. Por lo cual constituyen puntos críticos en la elaboración y deberán ser cuidadosamente controlados para evitar el deterioro del producto. Por otro lado, se concluyó que en las superficies limpias, antes que entren en contacto con los jugos de uva pre-concentrados o concentrados, no hubo incidencia de Z. rouxii. Este hecho sugiere que las prácticas sanitarias utilizadas en las tres plantas serían capaces de eliminar las poblaciones de Z. rouxii de las superficies, siempre y cuando los restos de mosto sean completamente removidos de todas las áreas en contacto con el producto. Siete especies de levaduras fueron identificadas en las superficies: Wickerhamomyces anomalus, Torulaspora delbrueckii, Citeromyces matritensis, Lachancea thermotolerans, Metschnikowia pulcherrima, Candida orthopsilosis y Candida apícola. El hallazgo de estas especies osmotolerantes, sugiere que las mismas presentan características que les permitieron persistir en las superficies higienizadas, los recuentos obtenidos fueron en muchos casos muy elevados. Estas especies han sido descritas como asociadas a los ambientes de las plantas elaboradoras de productos azucarados pero no han sido clasificadas como alterantes del producto per se.

Mycorrhizal preferences and fine spatial structure of the epiphytic orchid Epidendrum rhopalostele

• Premise of the study: The presence of compatible fungi is necessary for epiphytic orchid recruitment. Thus, identifying associated mycorrhizal fungi at the population level is essential for orchid conservation. Recruitment patterns may also be conditioned by factors such as seed dispersal range and specific environmental characteristics. • Methods: In a forest plot, all trees with a diameter at breast height >1 cm and all individuals of the epiphytic orchid Epidendrum rhopalostele were identified and mapped. Additionally, one flowering individual of E. rhopalostele per each host tree was randomly selected for root sampling and DNA extraction. • Key results: A total of 239 E. rhopalostele individuals were located in 25 of the 714 potential host trees. Light microscopy of sampled roots showed mycorrhizal fungi in 22 of the 25 sampled orchids. Phylogenetic analysis of ITS1-5.8S-ITS2 sequences yielded two Tulasnella clades. In four cases, plants were found to be associated with both clades. The difference between univariate and bivariate K functions was consistent with the random labeling null model at all spatial scales, indicating that trees hosting clades A and B of Tulasnella are not spatially segregated. The analysis of the inhomogenous K function showed that host trees are not clustered, suggesting no limitations to population-scale dispersal. χ2 analysis of contingency tables showed that E. rhopalostele is more frequent on dead trees than expected. • Conclusions: Epidendrum rhopalostele establishes mycorrhizal associations with at least two different Tulasnella species. The analysis of the distribution patterns of this orchid suggests a microsite preference for dead trees and no seed dispersal limitation.

Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in Saccharomyces cerevisiae

RPP2, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of Saccharomyces cerevisiae. Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P. Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells. Depletion of Rpp2 protein in vivo causes accumulation of precursor tRNAs with unprocessed introns and 5′ and 3′ termini, and leads to defects in the processing of the 35S precursor rRNA. Rpp2-depleted cells are defective in processing of the 5.8S rRNA. Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor rRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30. These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5′ end and E2 at the 3′ end of 18S rRNA, and E3 at or near the 5′ end of 5.8S rRNA.

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

A nucleolar protein related to ribosomal protein L7 is required for an early step in large ribosomal subunit biogenesis

The Saccharomyces cerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them. Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for cell growth. In this report, we show that Rlp7p is a nucleolar protein that plays a critical role in processing of precursors to the large ribosomal subunit RNAs. Pulse–chase labeling experiments with Rlp7p-depleted cells reveal that neither 5.8SS, 5.8SL, nor 25S is produced, indicating that both the major and minor processing pathways are affected. Analysis of processing intermediates by primer extension indicates that Rlp7p-depleted cells accumulate the 27SA3 precursor RNA, which is normally the major substrate (85%) used to produce the 5.8S and 25S rRNAs, and the ratio of 27SBL to 27SBS precursors changes from approximately 1:8 to 8:1 (depleted cells). Because 27SA3 is the direct precursor to 27SBS, we conclude that Rlp7p is specifically required for the 5′ to 3′ exonucleolytic trimming of the 27SA3 into the 27SBS precursor. As it is essential for processing in both the major and minor pathways, we propose that Rlp7p may act as a specificity factor that binds precursor rRNAs and tethers the enzymes that carry out the early 5′ to 3′ exonucleolytic reactions that generate the mature rRNAs. Rlp7p may also be required for the endonucleolytic cleavage in internal transcribed spacer 2 that separates the 5.8S rRNA from the 25S rRNA.

Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana

Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes RNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-O-methyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized that RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.