Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

Genetic diversity of 16S rRNA and mcrA genes from methanogenic archaeas

Methanogenic archaeas are found in aquatic and terrestrial environments and are fundamental in the conversion of organic matter into methane, a gas that has a potential use as renewable source of energy, which is also considered as one of the main agents of the greenhouse effect. The vast majority of microbial genomes can be identified by a conservative molecular marker, the 16S ribosomal gene. However, the mcrA gene have been using in studies of methanogenic archaea diversity as an alternative marker, highly conserved and present only in methanogens. This gene allows the expression of the enzyme Methyl-coenzyme M reductase, the main agent in converting by-products of anaerobic digestion into methane. In this context, we aimed to study the genetic diversity of mcrA and 16S rRNA genes sequences available in databases. The nucleotide sequences were selected from the NCBI. The heterozygosity and molecular diversity indexes were calculated using the Arlequin 3.5 software, with plots generated by package R v3.0. The diversity and heterozygosity indices for both genes may have been influenced by the number and size of the sequences. Descriptive analysis of genetic diversity generated by sequences deposited in databases allowed a detailed study of these molecules. It is known that the organisms in a population are genetically distinct, and that, despite having similarities in their gene composition, the differences are essential for their adaptation to different environments.

Genetic diversity of 16S rRNA and mcrA genes from methanogenic archaeas

Methanogenic archaeas are found in aquatic and terrestrial environments and are fundamental in the conversion of organic matter into methane, a gas that has a potential use as renewable source of energy, which is also considered as one of the main agents of the greenhouse effect. The vast majority of microbial genomes can be identified by a conservative molecular marker, the 16S ribosomal gene. However, the mcrA gene have been using in studies of methanogenic archaea diversity as an alternative marker, highly conserved and present only in methanogens. This gene allows the expression of the enzyme Methyl-coenzyme M reductase, the main agent in converting by-products of anaerobic digestion into methane. In this context, we aimed to study the genetic diversity of mcrA and 16S rRNA genes sequences available in databases. The nucleotide sequences were selected from the NCBI. The heterozygosity and molecular diversity indexes were calculated using the Arlequin 3.5 software, with plots generated by package R v3.0. The diversity and heterozygosity indices for both genes may have been influenced by the number and size of the sequences. Descriptive analysis of genetic diversity generated by sequences deposited in databases allowed a detailed study of these molecules. It is known that the organisms in a population are genetically distinct, and that, despite having similarities in their gene composition, the differences are essential for their adaptation to different environments.(AU)

Archaea diversity in vegetation gradients from the Brazilian Cerrado

We used 16S rRNA sequencing to assess the archaeal communities across a gradient of Cerrado. The archaeal communities differed across the gradient. Crenarcheota was the most abundant phyla, with Nitrosphaerales and NRPJ as the predominant classes. Euryachaeota was also found across the Cerrado gradient, including the classes Metanocellales and Methanomassiliicoccaceae.(AU)

The hyporheic zone and its functions: revision and research status in Neotropical regions / A zona hiporréica e as suas funções: revisão e estado da arte da pesquisa na região neotropical

The hyporheic zone (HZ), as the connecting ecotone between surface- and groundwater, is functionally part of both fluvial and groundwater ecosystems. Its hydrological, chemical, biological and metabolic features are specific of this zone, not belonging truly neither to surface- nor to groundwater. Exchanges of water, nutrients, and organic matter occur in response to variations in discharge and bed topography and porosity. Dynamic gradients exist at all scales and vary temporally. Across all scales, the functional significance of the HZ relates to its activity and connection with the surface stream. The HZ is a relatively rich environment and almost all invertebrate groups have colonized this habitat. This fauna, so-called hyporheos, is composed of species typical from interstitial environment, and also of benthic epigean and phreatic species. The hyporheic microbiocenose consists in bacteria, archaea, protozoa and fungi. The HZ provides several ecosystem services, playing a pivotal role in mediating exchange processes, including both matter and energy, between surface and subterranean ecosystems, functioning as regulator of water flow, benthic invertebrates refuge and place of storage, source and transformation of organic matter. The hyporheic zone is one of the most threatened aquatic environments, being strongly influenced by human activities, and the least protected by legislation worldwide. Its maintenance and conservation is compelling in order to preserve the ecological interconnectivity among the three spatial dimensions of the aquatic environment. Although several researchers addressed the importance of the hyporheic zone early, and most contemporary stream ecosystem models explicitly include it, very little is known about the HZ of Neotropical regions. From a biological standpoint, hyporheos fauna in Neotropical regions are still largely underestimated. This review focuses on a brief presentation of the hyporheic zone and its functions and significance as an ecotone. We also highlighted the key aspects considering also the current status of research in Neotropical regions.(AU)

A zona hiporréica, como ecótono de ligação entre a superfície e as águas subterrâneas, é parte funcional seja dos ecossistemas fluviais seja das águas subterrâneas. As características hidrológicas, as características químicas, biológicas e metabólicas são específicas desta zona, não pertencendo verdadeiramente nem a superfície nem às águas subterrâneas. Trocas de água, nutrientes e matéria orgânica ocorrem em resposta a variações na descarga, topografia do álveo e porosidade. Gradientes dinâmicos existem em todas as escalas e variam temporalmente. Em todas as escalas, o significado funcional da zona hyporheic relaciona-se com a sua conexão e atividades com a água superficial. O HZ é um ambiente relativamente rico e quase todos os grupos de invertebrados colonizaram este habitat. Esta fauna, chamada hyporheos, é composta por espécies típicas do ambiente intersticial, e também de espécies bentônicas epígeas e freáticas. A microbiocenose consiste em bactérias, arqueobactérias, fungos e protozoários. O HZ fornece vários serviços para o ecossistema, desempenhando um papel fundamental na mediação de processos de troca, incluindo seja a matéria, seja a energia, entre os ecossistemas superfíciais e os subterrâneos, funcionando como regulador do fluxo de água, de refúgio para invertebrados bentônicos e local de armazenagem, fonte e transformação de matéria orgânica. A zona hyporheic é um dos ambientes aquáticos mais ameaçados, sendo fortemente influenciado pelas atividades humanas, e um dos menos protegidos pela legislação em todo o mundo. A sua manutenção e conservação é necessaria para preservar a interconectividade ecológica entre as três dimensões espaciais do ambiente aquático. Apesar de vários pesquisadores aborem a importância da zona hyporheic a tempo, e a maioria dos modelos de ecossistemas atualmente incluí-lo de forma explicita, muito pouco se sabe sobre o HZ das regiões neotropicais. Do ponto de vista biológico, a fauna hiporréica das regiões neotropicais é ainda largamente subestimada. Esta revisão visa apresentar de forma resumida a zona hiporréica, suas funções e importância como ecótono. Também visa destacar os aspectos principais considerando também o estado actual da investigação em regiões neotropicais.(AU)

Large scale distribution of bacterial communities in the upper Paraná River floodplain

A bacterial community has a central role in nutrient cycle in aquatic habitats. Therefore, it is important to analyze how this community is distributed throughout different locations. Thirty-six different sites in the upper Paraná River floodplain were surveyed to determine the influence of environmental variable in bacterial community composition. The sites are classified as rivers, channels, and floodplain lakes connected or unconnected to the main river channel. The bacterial community structure was analyzed by fluorescent in situ hybridization (FISH) technique, based on frequency of the main domains Bacteria and Archaea, and subdivisions of the phylum Proteobacteria (Alpha-proteobacteria, Beta-proteobacteria, Gamma-proteobacteria) and the Cytophaga-Flavobacterium cluster. It has been demonstrated that the bacterial community differed in density and frequency of the studied groups. And these differences responded to distinct characteristics of the three main rivers of the floodplain as well as to the classification of the environments found in this floodplain. We conclude that dissimilarities in the bacterial community structure are related to environmental heterogeneity, and the limnological variables that most predicted bacterial communities in the upper Paraná River floodplain was total and ammoniacal nitrogen, orthophosphate and chlorophyll-a.