Biology and population ecology of uncultured Archaea in natural environments analyzed by taxon-specific molecular markers = Biología y ecología poblacional de Archaeas no cultivadas en ambientes naturales analizadas mediante marcadores moleculares específicos

Los microorganismos del Dominio Archaea son un componente común del plancton que ha pasado, mayoritariamente desapercibido para la Limnología durante mucho tiempo. Sólo tras la aplicación de técnicas moleculares en ecología microbiana se ha descubierto su enorme riqueza biológica y abundancia tanto en ambientes acuáticos como terrestres, más allá del grupo de las archaea metanógenas. Estudios recientes han demostrado que los hábitats de agua dulce tienen la mayor diversidad filogenética y representan entornos prometedores para el descubrimiento de nuevos linajes de Archaea. Uno de los ambientes que constituyen una fuente inexplorada de riqueza de nuevos filotipos de Archaea son los lagos alpinos, donde diferencias llamativas en la composición filogenética y abundancia de las arqueas se han reportado recientemente. Esto hace que los sistemas lacustres sean un modelo útil para comprender los factores ecológicos y la influencia de la heterogeneidad del hábitat sobre la composición de microorganismos Archaea, y para vincular los rasgos ecológicos y metabólicos de las arqueas con el funcionamiento del ecosistema. Esta tesis tiene como objetivo combinar microbiología ambiental, técnicas moleculares y aproximaciones bioinformáticas para progresar en el conocimiento de la distribución ecológica y ecofisiología de filotipos representativos de las arqueas de agua dulce superando la limitación de su baja cultivabilidad. Para este propósito, se diseñaron y optimizaron cebadores específicos para el gen SAGMGC-1 16S rRNA y los diferentes ecotipos de archaea oxidadoras de amonio, AOA (gen amoA), y se cuantifico mediante qPCR las distribuciones espacio-temporales a lo largo de una exploración anual en un lago profundo de alta montaña, donde las deposiciones atmosféricas son la principal fuente de nitrógeno reactivo. Adicionalmente se exploraron las diferencias espacio-temporales en el plancton superficial de un gran conjunto de lagos con gradientes ambientales marcados, mediante el diseño y optimización de cebadores específicos para el gen 16S rRNA de los grupos de euryarchaeota MEG y DSEG. Por último, se construyo una base de datos genética detallada del gen ribosómico 16S rRNA de Archaea utilizando SILVA y nuestros propios datos genéticos generados a partir de trabajos anteriores, a partir de esto se diseño y optimizo la sonda específica de CARDFISH para el grupo taumarchaeota SAGMGC-1, combinando aproximaciones in silico, in vitro e in situ. Posteriormente se cuantificó selectivamente dicha población a lo largo de un gradiente ambientalThe annual dynamics of three different ammonia-oxidizing archaea (AOA) ecotypes (amoA gene) and of the SAGMCG-1 (Nitrosotalea-like aquatic Thaumarchaeota) group (16S rRNA gene) were studied by newly designed specific primers and quantitative PCR analysis in a deep oligotrophic high mountain lake (Lake Redon, Limnological Observatory of the Pyrenees, Spain). We observed segregated distributions of the main AOA populations, peaking separately in time and space, and under different ammonia concentrations and irradiance conditions. Strong positive correlation in gene abundances was found along the annual survey between 16S rRNA SAGMCG-1 and one of the amoA ecotypes, suggesting the potential for ammonia oxidation in the freshwater SAGMCG-1 clade. We also observed dominance of Nitrosotalea-like ecotypes over Nitrosopumilus-like (MG 1.1a), and not the same annual dynamics for the two thaumarchaeotal clades. The fine scale segregation in space and time of the different AOA ecotypes indicated the presence of phylogenetically close but ecologically segregated AOA species specifically adapted to specific environmental conditions. It remains to be elucidated what would be such environmental drivers. Subsequently quantitative environmental distribution of two widely distributed uncultured freshwater Euryarchaeota with unknown functional role, was explored by newly designed qPCR primers targeting the 16S rRNA gene of clades MEG (Miscellaneous Euryarchaeota Group containing the groups pMC2A384 and VALII/Eury4) and DSEG (Deep-Sea Euryarchaeotal Groups, targeting the cluster named VALIII containing the DHVE-3/DSEG, BC07-2A-27/DSEG-3, and DSEG-2 groups), respectively. The summer surface plankton of 28 lakes was analyzed and one additional dimictic deep lake, Lake Redon, was temporally and vertically surveyed covering seasonal limnological variability. A trophic range between 0.2 and 5.2 μg/L Chl a, and pH span from 3.8 to 9.5 was explored at altitudes between 632 and 2590 m above sea level. The primers showed to be highly selective with c. 85% coverage and 100% specificity. Only pH significantly explained the changes observed in gene abundances and environment. In Lake Redon, DSEG bloomed in deep stratified waters both in summer and early spring, and MEG at intermediate depths during the ice-cover period. Overall, MEG and DSEG showed a differential ecological distribution although correlational analyses indicated lack of coupling of both euryarchaeota with phytoplankton (chlorophyll a). However, an intriguing positive and significant relationship was found between DSEG and putative ammonia oxidizing Thaumarchaeota. The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with the enzyme horseradish peroxidase (HRP), they can be used to identify single microbial cells directly by fluorescence in situ hybridization. Based on comparative analyses of 16S rRNA sequences we have located sites specific for the SAGMCG-1 group of Thaumarchaeota, including the species Nitrosotalea devanaterra. Oligonucleotides complementary to these signature regions were evaluated as potential nucleic acid probes for the differentiation of SAGMCG-1 lineage. Hybridization conditions were optimized by the addition of increasing formamide concentrations to the hybridization buffer. The fluorescence intensity was enhanced and the effects on the cell visualization were improved by design of two adjacent helpers for the probe. CARD-FISH hybridization using N. devanaterra demonstrated high probe specificity in vitro, and whole cell hybridization with fluorescent probe derivatives allowed the identification and quantification in selected soil samples of individual microbial cells in situ. The probes will be useful for determinative studies and for the in situ monitoring of population distribution and dynamics in microbial communities closely related to the N biogeochemical cycling