A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract

Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt

Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPBFP7 Ideas: European Research Council, Grant/Award Number: ERC Advanced Grant #250-35

Ecología microbiana de bacterias del suelo y su mecanismo de acción en el desarrollo de "Solanum lycopersicum"

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-06-2007In agricultural soils, due to intensive practices, there are lost of nutrients and damage of soil structure, which produce a reduction in soil fertility and in harvest yield. To stop these effects, traditionally, it have been employed chemical fertilizers and manure. The use of microorganisms (plant growth promoting rhizobacteria) in agriculture as an alternative to traditional methods is just starting, because so far there is ignorance about their mechanisms of action in the plant growth and development, and because the distrust about the real effectiveness. But the need of alternative method to tradicional ones which does not damage human or animal health, and the environment is an interesting field in development. Today there is more information about the possible mechanisms of action of these microorganisms as fertilizers and protectors against soilborne phytopatogens. This work is a first approach to understand the way by which some of these microorganisms act in plant growth promotion and upon plant defense mechanisms with the aims of their possible use in agriculture, trying to vanish the general distrust about their use as a real alternative to chemical fertilizers and current environmentally non safe methods. The results of this thesis are grouped in 3 sections, the first about microbial ecology in which we try to see the bacterial behavior in the natural environment. The second section deals with the study of the bacterial effect on plant growth promotion, based on structural and molecular approaches. And the third section is a phylogenetic study of Beijerinckiaceae family focused on Beijerinckia genus, specifically on Beijerinckia fluminensis, because the lack of information about this species since its isolation and identification by Johanna Döbereiner in 1958

Beijerinckia doebereinerae sp. nov.

17 pages.Reclassification of Beijerinckia fluminensis CIP 106281T and Beijerinckia fluminensis UQM 1685T as Rhizobium radiobacter strains, and proposal of Beijerinckia fluminensis LMG 2819 as Beijerinckia doebereinerae sp. nov.Peer reviewe

Fungal iron biomineralization in río tinto

Although there are many studies on biomineralization processes, most of them focus on the role of prokaryotes. As fungi play an important role in different geological and biogeochemical processes, it was considered of interest to evaluate their role in a natural extreme acidic environment, Río Tinto, which has a high level of fungal diversity and a high concentration of metals. In this work we report, for the first time, the generation of iron oxyhydroxide minerals by the fungal community in a specific location of the Tinto basin. Using Transmission Electron Microscopy (TEM) and High Angle Angular Dark Field coupled with Scanning Transmission Electron Microscopy (HAADF-STEM) and Energy-Dispersive X-ray Spectroscopy (EDX), we observed fungal structures involved in the formation of iron oxyhydroxide minerals in mineralized sediment samples from the Río Tinto basin. Although Río Tinto waters are supersaturated in these minerals, they do not precipitate due to their slow precipitation kinetics. The presence of fungi, which simply provide charged surfaces for metal binding, favors the precipitation of Fe oxyhydroxides by overcoming these kinetic barriers. These results prove that the fungal community of Río Tinto participates very actively in the geochemical processes that take place there.European Research Council Advanced Grant ERC-250350/IPBSLPeer Reviewe

The deep continental subsurface: the dark biosphere

Although information from devoted geomicrobiological drilling studies is limited, it is clear that the results obtained so far call for a systematic exploration of the deep continental subsurface, similar to what has been accomplished in recent years by the Ocean Drilling Initiatives. In addition to devoted drillings from the surface, much of the continental subsurface data has been obtained using different subterranean “windows,” each with their correspondent limitations. In general, the number and diversity of microorganisms decrease with depth, and the abundance of Bacteria is superior to Archaea. Within Bacteria, the most commonly detected phyla correspond to Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Within Archaea, methanogens are recurrently detected in most analyzed subsurface samples. One of the most controversial topics in the study of subsurface environments is whether the available energy source is endogenous or partly dependent on products photosynthetically generated in the subsurface. More information, at better depth resolution, is needed to build up the catalog of deep subsurface microbiota and the biologically available electron acceptors and donors.Ministerio de Economía y Competitividad, grant CGL2015-66242-RPeer Reviewe

Active microbial biofilms in deep poor porous continental subsurface rocks

Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments.MINECOPeer Reviewe

A micromorphological and phylogenetic study of Sarcocornia A.J. Scott (Chenopodiaceae) on the Iberian Peninsula

This study presents a comprehensive revision of the genus Sarcocornia (Chenopodiaceae) on the Iberian Peninsula based on macromorphological, micromorphological and phylogenetic data, and considering caryological, ecological and biogeographical information. Three species of Sarcocornia have been identified on the Iberian Peninsula: Sarcocornia perennis (Miller) A.J. Scott, Sarcocornia fruticosa (L.) A.J. Scott and Sarcocornia alpini (Lag.) Rivas-Martínez. Several authors have proposed that S. alpini is a specific and subspecific rank of S. perennis. Fuente, Rufo and Sánchez-Mata have recently described a new species, Sarcocornia hispanica. The micromorphological and molecular studies (sequence of the internal transcribed spacer region) indicate that there is a broad diversity within Sarcocornia in the Western Mediterranean. This article proposes a new species (Sarcocornia pruinosa) and subspecies (S. alpini subsp. carinata) in view of the new data.Ministerio de Educación y Ciencia (CGL2009-11059, CTM2010-18456); Ministerio de Ciencia e InnnovaciónPeer Reviewe

A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract