Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Nop53p interacts with 5.8S rRNA co-transcriptionally, and regulates processing of pre-rRNA by the exosome

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Delta nop53/GAL:NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis

Background and Objective: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. Material and Methods: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. Results: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. Conclusion: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Phylogenetic Validation of the Genera Angomonas and Strigomonas of Trypanosomatids Harboring Bacterial Endosymbionts with the Description of New Species of Trypanosomatids and of Proteobacterial Symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia cuiicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history. (C) 2011 Elsevier GmbH. All rights reserved.

Phylogenetic Analyses Based on Small Subunit rRNA and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

Utp25p, a nucleolar Saccharomyces cerevisiae protein, interacts with U3 snoRNP subunits and affects processing of the 35S pre-rRNA

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages of 35S pre-rRNA processing and give rise to the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors. The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis. Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation. Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels. Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation.

Sdo1p, the yeast orthologue of Shwachman-Bodian-Diamond syndrome protein, binds RNA and interacts with nuclear rRNA-processing factors

The Shwachman-Bodian-Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre-60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co-immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright (C) 2009 John Wiley & Sons, Ltd.

Genetic diversity and translocation in the marron, Cherax tenuimanus (Smith): implications for management and conservation

Approximately 440 base pairs (bp) of the mitochondrial 16S ribosomal ribose nucleic acid (rRNA) coding region were sequenced from 13 marron, Cherax tenuimanus (Smith), samples from six locations and samples of two additional Cherax species from Western Australia. The results indicated that, with the exception of the Margaret River, no variation was found within or between marron populations. In contrast, marron from the Margaret River were found to be polymorphic for two divergent haplotypes (2.76% divergence). These findings were consistent with allozyme data that highlight the general lack of genetic variability within and between populations of this species apart from the Margaret River stocks. The genetic polymorphisms in the Margaret River stocks contrasted with earlier studies and indicated the recent translocation and mixing of genetically differentiated stocks within this river system. The implications of these findings for the conservation and management of genetic diversity within marron are discussed.

Molecular taxonomy and phylogenetics of some species of Australian palaemonid shrimps

The evolutionary history and classification of the palaemonid shrimps has been the subject of constant speculation and debate. At present, all major systematic treatments have been based on morphological characteristics. To help resolve the phylogenetic relationships, and thus enable the creation of a classification system that reflects evolutionary history, a region of the 16S mitochondrial rRNA gene was sequenced for a number of Australian Palaemonidae. The resulting phylogenetic analyses indicated the presence of major anomalies in the current classification of Australian Palaemonidae. Significantly, three species belonging to three separate genera, Macrobrachium intermedium, Palaemon serenus, and Palaemonetes australis, are closely related, with genetic differences more characteristic with that of congeneric species. The results also demonstrate non-monophyly in Australian palaemonids with respect to both Palaemonetes and Macrobrachium.

Mitochondrial 12S rRNA sequences support the existence of a third species of freshwater blackfish (Percicthyidae: Gadopsis) from south-eastern Australia

Fish of the genus Gadopsis are a distinctive component of the freshwater fish fauna of south-eastern Australia. Gadopsis marmoratus and G. bispinosus are the only two species recognised within the genus, with the former of uncertain taxonomic status, as it is thought to be composed of at least two distinct geographical forms based on morphological and allozyme data. The objective of this study was to investigate DNA sequence divergence in Gadopsis, especially in the western portion of its distribution, using an approximately 400 base pair fragment of the mitochondrial small subunit 12S rRNA gene region in order to reassess the taxonomy of the genus. Individuals from 11 locations were sequenced and confirm that G. marmoratus and G. bispinosus are genetically distinct, and further that the G. marmoratus complex consists of two divergent clades representing the previously identified northern and southern forms. The degree of divergence between the three Gadopsis clades was similar (5–6% nucleotide substitutions), suggesting that they diverged from a common ancestor at approximately the same period in geological time. These results are consistent with previous allozyme studies and highlight the usefulness of mitochondrial DNA data coupled with allozyme information for clarifying taxonomic boundaries in morphologically conservative aquatic organisms.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda:Parastacidae) inferred from four mitochondrial gene regions

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

Discriminação de ambientes aquáticos poluídos e não poluídos através da amplificação e clivagem do rDNA 16S

O crescimento desordenado da população e a expansão mal planejada das cidades, tem levado a precariedade das condições de saneamento e abastecimento hídrico, tornando a água um importante meio de desenvolvimento e transporte de microrganismos, muitos destes patogênicos. Desta maneira, torna-se necessário além da busca de soluções, o monitoramento eficiente e constante dos níveis e focos de contaminação. O presente trabalho teve como objetivo principal diferenciar ambientes aquáticos poluídos de não poluídos por contaminação de origem fecal, através de padrões moleculares. Para tanto foram escolhidos os arroios Feijó e Carvão, localizados na região metropolitana de Porto Alegre, Rio Grande do Sul, de onde foram coletadas amostras de água no verão e no inverno de 2001, submetidas a determinação do número mais provável (NMP) de coliformes totais e fecais, através da fermentação em tubos múltiplos. No Arroio Feijó foram observados índices de coliformes fecais superiores ao arroio Carvão, portanto o primeiro foi considerado modelo de ambiente aquático poluído e o segundo, não poluído. Seguiu-se a extração do DNA total das amostras brutas, amplificação do rDNA 16S pela PCR e clivagem destes com as enzimas de restrição Rsa I, Taq I e Alu I. Os padrões de clivagens observados com Rsa I sugerem a discriminação destes ambientes, uma variação sazonal foi observada entre as amostras de verão e inverno. O método mostrou-se sensível para uma análise comparativa entre estruturas de comunidades bacterianas em ambientes aquáticos.

Bacterial functional redundancy along a soil reclamation gradient

A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU), BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site, the number and diversity of bacterial groups responding to four carbon substrates (L-serine, L-threonine, sodium citrate, and or-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.