Vida microbiana a temperaturas elevadas. Diversidad, aislamiento, termoestabilidad molecular y genómica

A pesar de los numerosos estudios publicados sobre la vida a altas temperaturas, aún existen aspectos fundamentales por comprender. La presente tesis estudia la vida microbiana a altas temperaturas desde diferentes enfoques. Por un lado, analizamos la influencia de la temperatura sobre la distribución de los microorganismos tomando como modelo un gradiente de temperatura natural de 50ºC en una fuente termal. La temperatura y las interacciones entre sus componentes resultaron ser determinantes para estructurar las comunidades microbianas. El aislamiento de una nueva especie de bacteria termófila extrema y anaeróbica, Fervidobacterium thailandense FC2004T, y la secuenciación de su genoma hace posible un estudio en detalle y su clasificación taxonómica. La comparación de cuatro genomas dentro del género Fervidobacterium ha permitido determinar que los genes que codifican transposasas son un modelo adecuado para analizar fenómenos de intercambio de material genético. (transferencia horizontal de genes, HGT) y evaluar la plasticidad de estos genomas y su historia evolutiva. Los microorganismos termófilos viven a temperaturas a las que numerosas biomoléculas, como el NADH, son inestables. La viscosidad influye decisivamente en la estabilización de esas pequeñas biomoléculas. La viscosidad intracelular en microorganismos termófilos (entre 50 y 80ºC) era relativamente elevada, lo que confirma el efecto estabilizador de la viscosidad. Esta tesis contribuye a comprender la importancia de la temperatura sobre los microorganismos y sus comunidades microbianas, su capacidad para desarrollarse a temperaturas elevadas, así como el dinamismo de sus genomas y fenotipos

Microbial divergence and evolution. The case of anammox bacteria

Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future

Cellular Viscosity in Prokaryotes and Thermal Stability of Low Molecular Weight Biomolecules

8 páginas.-- 6 figuras.-- 32 referenciasSome low molecular weight biomolecules, i.e., NAD(P)H, are unstable at high temperatures. The use of these biomolecules by thermophilic microorganisms has been scarcely analyzed. Herein, NADH stability has been studied at different temperatures and viscosities. NADH decay increased at increasing temperatures. At increasing viscosities, NADH decay rates decreased. Thus, maintaining relatively high cellular viscosity in cells could result in increased stability of low molecular weight biomolecules (i.e., NADH) at high temperatures, unlike what was previously deduced from studies in diluted water solutions. Cellular viscosity was determined using a fluorescent molecular rotor in various prokaryotes covering the range from 10 to 100°C. Some mesophiles showed the capability of changing cellular viscosity depending on growth temperature. Thermophiles and extreme thermophiles presented a relatively high cellular viscosity, suggesting this strategy as a reasonable mechanism to thrive under these high temperatures. Results substantiate the capability of thermophiles and extreme thermophiles (growth range 50–80°C) to stabilize and use generally considered unstable, universal low molecular weight biomolecules. In addition, this study represents a first report, to our knowledge, on cellular viscosity measurements in prokaryotes and it shows the dependency of prokaryotic cellular viscosity on species and growth temperature.The authors acknowledge funding from projects No. CSD2009-0006 and No. CGL2014-58762-P from the Spanish Ministry of Economy and Competitiveness and grants No. BIO-288 and No. RNM2529 from the Andalusian Government. Federal funds cofinanced these projects. Funding from the mobility program No. 003-ABEL-CM-2013 (NILS Science and Sustainability program, EEA grants) is also acknowledged.Peer Reviewe

Procedimiento de estabilización de biomoléculas

La presente invención se refiere a un procedimiento para la estabilización de biomoléculas caracterizado porque dicha estabilización se consigue manteniendo la viscosidad del medio en el cual se encuentran dichas biomoléculas. Es de aplicación a un amplio número de biomoléculas tales como ATP o NADH y permite la utilización de las mismas en procedimientos analíticos, clínicos o médicos que se vayan a llevar a cabo en condiciones no adecuadas para el mantenimiento de su estabilidad.Peer reviewedConsejo Superior de Investigaciones CientíficasB1 Patente sin examen previ

Fervidobacterium thailandense sp. nov., an extremely thermophilic bacterium isolated from a hot spring

4 páginas.-- 2 figuras.-- 19 referencias.-- Two supplementary tables and four supplementary figures are available with the online Supplementary Material.Strain FC2004T, a strictly anaerobic, extremely thermophilic heterotroph, was isolated from a hot spring in Thailand. Typical cells of strain FC2004T were rod shaped (0.5–0.6×1.1–2.5 µm) with an outer membrane swelling out over an end. Filaments (10–30 µm long) and membrane-bound spheroids containing two or more cells inside (3–8 µm in diameter) were observed. The temperature range for growth was 60–88°C (optimum 78–80°C), pH range was 6.5–8.5 (optimum pH 7.5) and NaCl concentration range was 0 to <5 g l−1 (optimum 0.5 g l−1). S0 stimulated growth yield. S2O3 2– and NO3 − did not influence growth. Glucose, maltose, sucrose, fructose, cellobiose, CM-cellulose and starch were utilized for growth. The membrane was composed mainly of the saturated fatty acids C16:0 and C18:0. The DNA G+C content was 45.8 mol%. The 16S rRNA gene sequence of strain FC2004T revealed highest similarity to species of the genus Fervidobacterium : F. pennivorans DSM 9078T (97–96 %), F. islandicum AW-1 (96 %), F. changbaicum CBS-1T (96 %), F. islandicum H21T (95 %), F. nodosum Rt17-B1T (95 %), F. riparium 1445tT (95 %) and F. gondwanense AB39T (93 %). Phylogenetic analysis of 16S rRNA gene sequences and average nucleotide identity analysis suggested that strain FC2004T represented a novel species within the genus Fervidobacterium , for which the name Fervidobacterium thailandense sp. nov. is proposed. The type strain is FC2004T (=JCM 18757T=ATCC BAA-2483T).Peer reviewe

Cellular Viscosity in Prokaryotes and Thermal Stability of Low Molecular Weight Biomolecules

Prepint, Texto y GráficosSome low molecular weight biomolecules, i.e., NAD(P)H, are unstable at high temperatures. The use of these biomolecules by thermophilic microorganisms has been scarcely analyzed. Herein, NADH stability has been studied at different temperatures and viscosities. NADH decay increased at increasing temperatures. At increasing viscosities, NADH decay rates decreased. Thus, maintaining relatively high cellular viscosity in cells could result in increased stability of low molecular weight biomolecules (i.e., NADH) at high temperatures, unlike what was previously deduced from studies in diluted water solutions. Cellular viscosity was determined using a fluorescent molecular rotor in various prokaryotes covering the range from 10 to 100°C. Some mesophiles showed the capability of changing cellular viscosity depending on growth temperature. Thermophiles and extreme thermophiles presented a relatively high cellular viscosity, suggesting this strategy as a reasonable mechanism to thrive under these high temperatures. Results substantiate the capability of thermophiles and extreme thermophiles (growth range 50–80°C) to stabilize and use generally considered unstable, universal low molecular weight biomolecules. In addition, this study represents a first report, to our knowledge, on cellular viscosity measurements in prokaryotes and it shows the dependency of prokaryotic cellular viscosity on species and growth temperature.España Mineco CSD2009-0006 CGL2014-58762-PJunta de Andalucía BIO-288 RNM2529NILS NILS 003-ABEL-CM-201

Evidence of horizontal gene transfer by transposase gene analyses in Fervidobacterium species

21 páginas.-- 12 figuras.-- 5 tablas.--31 referenciasHorizontal Gene Transfer (HGT) plays an important role in the physiology and evolution of microorganisms above all thermophilic prokaryotes. Some members of the Phylum Thermotogae (i.e., Thermotoga spp.) have been reported to present genomes constituted by a mosaic of genes from a variety of origins. This study presents a novel approach to search on the potential plasticity of Fervidobacterium genomes using putative transposase-encoding genes as the target of analysis. Transposases are key proteins involved in genomic DNA rearrangements. A comprehensive comparative analysis, including phylogeny, non-metric multidimensional scaling analysis of tetranucleotide frequencies, repetitive flanking sequences and divergence estimates, was performed on the transposase genes detected in four Fervidobacterium genomes: F. nodosum, F. pennivorans, F. islandicum and a new isolate (Fervidobacterium sp. FC2004). Transposase sequences were classified in different groups by their degree of similarity. The different methods used in this study pointed that over half of the transposase genes represented putative HGT events with closest relative sequences within the phylum Firmicutes, being Caldicellulosiruptor the genus showing highest gene sequence proximity. These results confirmed a direct evolutionary relationship through HGT between specific Fervidobacterium species and thermophilic Firmicutes leading to potential gene sequence and functionality sharing to thrive under similar environmental conditions. Transposase-encoding genes represent suitable targets to approach the plasticity and potential mosaicism of bacterial genomes.This study was supported by funding through projects from the Spanish Ministry of Economy and Competitiveness (CSD2009-00006 and CGL2014-58762-P) and the Regional Government of Andalusia (RNM2529 and BIO288). The participation of FEDER funds is acknowledgedPeer Reviewe

Viscosidad intracelular en Procariotas

El citoplasma es un sistema altamente complejo donde se lleva a cabo el metabolismo celular. Se considera que el citoplasma presenta una consistencia similar a un gel más que a una solución acuosa diluida. Esto podría influir el comportamiento de distintas biomoléculas. La viscosidad del citoplasma en eucariotas se ha determinado recientemente1 pero no así en procariotas. Conocer la viscosidad intracelular es necesario para analizar el comportamiento de numerosas biomoléculas. La viscosidad se reduce exponencialmente al aumentar la temperatura lo que conlleva consecuencias críticas para el mantenimiento del metabolismo celular, por ejemplo en termófilos. Previamente2 hemos citado el efecto estabilizador que presenta la viscosidad para diversas biomoléculas de pequeño tamaño (por ejemplo, NADH, ATP, etc.). Por tanto, es importante determinar la viscosidad intracelular para estudiar la posible estabilización de biomoléculas en condiciones de baja o alta temperatura y evaluar la capacidad de procariotas para responder a cambios de temperatura. Utilizando un rotor molecular fluorescente (RY3)1,3 se determinó la viscosidad intracelular de distintos procariotas desde 10ºC a 100ºC. Se analizaron las bacterias Escherichia coli, Pseudomonas aeruginosa, Lactococcus lactis lactis, Geobacillus thermoglucosidasius, Fervidobacterium thailandense y la arquea Pyrococcus furiosus. Las cubiertas celulares son permeables a RY3 por lo que la viscosidad intracelular puede determinarse a partir de medidas de fluorescencia realizadas por espectrofluorometría y por FLIM (Fluorescence Lifetime Imaging)3. Algunas bacterias son capaces de regular su viscosidad intracelular en función de la temperatura ambiental, como Lactococcus. Las bacterias parecen presentar una viscosidad relativamente elevada (superior al agua) aunque Escherichia y Pseudomonas eran excepciones con viscosidad similar al agua. Pyrococcus indicaba que debido a que la viscosidad varía mínimamente por encima de 80ºC, cambios de viscosidad celular no serían eficientes. Estos resultados confirman que la regulación de la viscosidad intracelular puede ser un mecanismo para el mantenimiento de las funciones celulares a alta y baja temperatura, incluyendo la estabilización de pequeñas biomoléculas hasta aproximadamente 80ºC. Bibliografía: 1 Peng, X et al. 2011. Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity. Ap J Am Chem Soc 133: 6626-6635 2 Cuecas, A, Cruces, J, Portillo, MC, Gonzalez, JM. 2013. Viscosity as a factor controlling thermostability of low-molecular weight biomolecules at elevated temperatures. RedEx 2013. Granada 3 Cuecas, A, Cruces, J, Galisteo-López, JF, Peng, X, Gonzalez, JM. 2016. Cellular viscosity in prokaryotes and thermal stability of low molecular weight biomolecules. Biophys J 111: 875-882Proyecto Junta de Andalucia BIO288Peer Reviewe

Evidence of directed horizontal gene transfer by transposase gene analyses in Fervidobacterium species.

Poster presentación en el 10th International Congress on Extremophiles (Extremophiles 2014), September 7-11, 2014, Saint Petersburg, RussiaHorizontal Gene Transfer events play an important role in the physiology and evolution of microorganisms above all thermophilic prokaryotes. Some members of the phylum Thermotogae (e. g., Thermotoga spp.) have been reported to present genomes constituted by a mosaic of genes from a variety of origins. This study presents a novel approach to search on the potential plasticity of Fervidobacterium genomes using putative transposase-encoding genes as the target genes of analysis. Transposases are key proteins involved in genomic DNA rearrangements within a genome and transferences between organisms, including different species from distantly related taxonomic categories. A comprehensive genome comparative analysis, including phylogeny, codon usage, and %GC content, is being performed for the transposases and related sequences present in three Fervidobacterium species: F. nodosum, F. pennivorans and including a newly reported isolate (candidate F. thailandensis). These transposases could be classified in different groups attending to their level of similarity. Only a low fraction of these sequences showed high similarity to transposase sequences represented in members of the phylum Thermotogae. Interestingly, sequence phylogeny, codon usage and %GC content showed that most groups of transposases were closely related to transposase sequences from different representatives of the Firmicutes Phylum. Other taxonomic categories, such as the phylum Nitrospirae and the Domain Archaea, showed relationship to some transposase genes in Fervidobacterium species but at a lower extent. This information suggests a direct evolutive relationship through horizontal gene transfer between specific thermophilic Firmicutes and Fervidobacterium species which leads to sharing genes, genome fragments and potential functionality besides putative environmental habitats.Peer Reviewe

Evidencia de transferencia horizontal de genes mediante el estudio de las transposasas en el género Fervidobacterium

Comunicación oral presentada en la XII Reunión Red Nacional Organismos Extremófilos. (REDEX2014) 031 Sesión 4 : Metagenómica y Fisiología, pag. 42. Libro de resumenes.Los fenómenos de transferencia horizontal de genes (HGT) juegan un papel relevante en la fisiología y la evolución de los microorganismos, especialmente en procariotas termófilos. Los genomas de algunos miembros del phylum Thermotogae (por ejemplo, Thermotoga spp.) se proponen como un mosaico formado por genes con diversos orígenes. Las transposasas son proteínas claves que intervienen en reordenamientos de ADN genómico dentro de un genoma, así como en transferencias de material genético entre organismos, incluyendo diferentes especies de categorías taxonómicas alejadas. Este estudio presenta un nuevo enfoque para detectar procesos de HGT mediante el estudio de los genes que codifican para transposasas en genomas del género Fervidobacterium. Para ello, se han llevado a cabo análisis filogenéticos, multivariables de frecuencia de uso de codones y de frecuencia de tetranucleótidos, así como la determinación y comparación de las secuencias repetitivas invertidas asociadas a las transposasas de tres especies: F. nodosum, F. pennivorans y una nueva cepa, Fervidobacterium sp. FC2004, recientemente aislada en nuestro laboratorio. Las transposasas detectadas se clasificaron en diferentes grupos atendiendo a su grado de similitud. Los análisis filogenéticos, multivariables y la comparación entre secuencias repetitivas invertidas coincidían en distinguir varios grupos de transposasas como indicadores de posibles fenómenos de HGT. Estos casos de HGT se muestran estrechamente relacionados con transposasas de diferentes representantes del phylum Firmicutes, siendo Caldicellulosiruptor uno de los géneros con representantes más cercanos en varios de los grupos estudiados. Los resultados obtenidos sugieren la existencia de varios fenómenos de HGT entre Fervidobacterium spp. y Firmicutes termófilos que se supone que ha llevado a estos grupos a facilitar su adaptación a las distintas condiciones existentes en sus ambientes extremos que frecuentemente cohabitan.Financiación: MICROGEN (Consolider CSD2009-00006; Ministerio de Economía y Competitividad) y BIO288 (Junta de Andalucía), ambos cofinanciados con fondos FEDER.Peer Reviewe