Connecting biodiversity and biogeochemical role by microbial metagenomics

[eng] The main objective of this PhD dissertation was to unveil the links between biogeochemistry and microbial diversity using metagenomics functional potential as a proxy to connect a mechanistic perspective with whole-system ecology. In order to explore the potential and limitations of metagenomics, two contrasted and widely studied lacustrine ecosystems with global ecological interest were selected. First, the Banyoles karstic system, a mirror to the past sulfurous and anoxic (euxinic) conditions where three lakes provide different euxinic situations where reduced compounds and sulfate were highly available: Lake Cisó, a small eutrophic lake with high carbon inputs, permanent anoxia and high euxinia; Lake Vilar, a meromictic mesotrophic lake; and Lake Banyoles basin CIII, a meromictic deep oligotrophic basin. And second, the ultraoligotrophic Lake Redon under three different limnological situations: the slush, an oxygenated environment with labile carbon from algal exudates; the epilimnion oxygenated layer with low availability of reduced compounds; and the hypolimnion, a dark layer under the influence of the mineralization microbial processes from the sediment. The previous knowledge on the biogeochemistry, ecology and functioning of these ecosystems, offered an excellent framework to connect the biodiversity and biogeochemical role by microbial metagenomics. The results showed higher bacterial novelty in the hypolimnia of euxinic lakes with Elusimicrobia and Chloroflexi harbouring the highest number of novel 16S rRNA gene sequences. The potential for dark carbon fixation in these lakes was tentatively assigned to Hydrogenophilales (Thiobacillus-like) and Gallionellales (Syderoxidans-like) via the Calvin cycle, Bacteroidales, Campylobacterales and Desulfarculales through the Arnon cycle, and Desulfobacterales via the reductive acetil-CoA cycle. If the findings from sulfurous lakes are equivalent to ancient oceans, Gallionellales may have played an important role in the biogeochemistry of the iron cycle, a low contribution of nitrification and archaea was probably accounting for the nitrogen cycle, Campylobacterales may have acted as the main players of denitrification, and Bacteroidales may have been main players for dissimilatory nitrate reduction to ammonium mainly in organic carbon-rich zones. The acquisition of laterally transferred genes offering new functional alternatives, was key in order to explain the ecological success of the recurrent green sulfur bacteria bloom in Lake Banyoles basin CIII. The drivers for such horizontal gene transfer were potentially phages. In the slush of the high-mountain Lake Redon, the community composition shift from winter to spring was characterized by a decrease in bacterial diversity, dominance of Bacteroidetes and consistent increases in bacterial abundance and production. The potential for nitrification in Lake Redon was mainly found in the slush and hypolimnion by ammonia-oxidizing bacteria. Lower abundances of nitrification genes were found in the epilimnion. Planktonic microbial communities of Lake Redon showed a higher genomic potential for assimilatory pathways (nitrite, phosphate and sulfate) than those from the Banyoles area. Overall, our analyses provide a new view on the well-known biogeochemical functioning of karstic and alpine lakes. Even with the limitations of metagenomics and annotation, we used the previous knowledge on the ecology of the lakes to prove that metagenomics is a very accurate way to unveil the links between functional potential and microbial biodiversity of any given ecosystem. We identified bacterial populations as potential key stone species of biogeochemical processes and provided new hypothesis to be further tested both through experimental approaches and detailed quantification of matter and energy fluxes.[cat] L'objectiu principal d'aquesta tesi és explorar els vincles entre la biogeoquímica i la diversitat microbiana, mitjançant la metagenòmica com a eina per determinar el potencial funcional de les comunitats i connectar la perspectiva mecanicista amb l’holística en l'estudi dels ecosistemes. Per tal d'explorar el potencial i les limitacions de la metagenòmica, es van seleccionar dos ecosistemes lacustres contrastats i àmpliament estudiats amb interès ecològic global. En primer lloc, el sistema càrstic de Banyoles, un equivalent de les condicions sulfuroses i anòxiques (euxíniques) en l'inici de la vida a la Terra, seleccionant tres llacs que proporcionen diferents situacions limnològiques amb alta disponibilitat de compostos reduïts i sulfat: el Llac Cisó , un petit llac eutròfic amb importants entrades de carboni orgànic, anòxia permanent i alta euxinia; el Llac Vilar, un llac mesotròfic i meromíctic; i la cubeta CIII del llac Banyoles, una cubeta profunda, oligotròfica i meromíctica. I en segon lloc, l’ultraoligotròfic Llac Redon en tres situacions limnològiques diferents: les capes de granissat (slush), un ambient oxigenat amb carboni làbil procedent d’exsudats d'algues; la massa d'aigua oxigenada de l’epilímnion amb baixa disponibilitat de compostos reduïts; i l'hipolímnion, una massa d'aigua en foscor sota la influència de processos microbians de mineralització que es donen en el sediment. El coneixement previ de la biogeoquímica, ecologia i funcionament d'aquests ecosistemes, ha ofert un excel·lent marc per a establir els vincles entre biodiversitat microbiana i funció biogeoquímica mitjançant una aproximació metagenòmica. En conjunt, aquesta tesi proporciona una nova visió sobre el funcionament biogeoquímic de llacs càrstics i alpins. Tot i les limitacions intrínseques a la metagenòmica i al procés d'anotació gènica, hem utilitzat els coneixements previs sobre l'ecologia dels llacs per demostrar que la metagenòmica és una eina molt precisa per donar a conèixer els vincles entre el potencial funcional i la biodiversitat microbiana de qualsevol ecosistema. S'han identificat les poblacions bacterianes potencialment responsables dels processos biogeoquímics i es proporcionen noves hipòtesis que hauran de ser comprovades mitjançant enfocaments experimentals i de quantificació dels fluxos de matèria i energia implicats

Taxonomy and functional interactions in upper and bottom waters of an oligotrophic high-mountain deep lake (Redon, Pyrenees) unveiled by microbial metagenomics

High mountain lakes are, in general, highly sensitive systems to external forcing and good sentinels of global environmental changes. For a better understanding of internal lake processes, we examined microbial biodiversity and potential biogeochemical interactions in the oligotrophic deep high-mountain Lake Redon (Pyrenees, 2240m altitude) using shotgun metagenomics. We analyzed the two ends of the range of environmental conditions found in Lake Redon, at 2 and 60 m depths. Bacteria were the most abundant component of the metagenomic reads (> 90%) and the diversity indices of both taxonomic (16S and 18S rRNA) and functional (carbon-, nitrogen-, sulfur-, and phosphorous-cycling) related genes were higher in the bottom dark layer than in the upper compartment. A marked segregation was observed both in biodiversity and in the dominant energy and biomass generating pathways between the extremes. The aerobic respiration was mainly dominated by heterotrophic Burkholderiales at the top and Actinobacteria and Burkholderiales at the lake bottom. The potential for an active nitrogen cycle (nitrogen fixation, nitrification, nitrite oxidation, and nitrate reduction) was mainly found at 60 m, and potential for methanogenesis, anaerobic ammonia oxidation and dissimilatory sulfur pathways were only observed there. Some unexpected and mostly unseen energy and biomass pathways were found relevant for the biogeochemical cycling in lake Redon, i.e., those related to carbon monoxide oxidation and phosphonates processing. We provide a general scheme of the main biogeochemical processes that may operate in the sentinel deep Lake Redon. This framework may help for a better understanding of the whole lake metabolism.This research was funded by Grants INTERACTOMA RTI2018-101205-BI00 to EOC and TRANSFER CGL2016-80124-C2-1-P to JC from the Spanish Office of Science (MICIU/AEI/MINECO) and European funding (ERDF/FEDER).Peer reviewe

Winter to spring changes in the slush bacterial community composition of a high mountain lake (Lake Redon, Pyrenees)

7 páginas, 4 figuras, 1 tabla.Bacterial community composition was analysed in the slush layers of snow-covered Lake Redon (2240 m altitude, Limnological Observatory of the Pyrenees, LOOP, NE Spain) in winter and spring and compared with bacteria from the lake water column, using 16S rRNA gene clone libraries and CARD-FISH counts. The set of biological data was related to changes in bacterial production and to other relevant environmental variables measured in situ. In winter, up to 70% of the 16S rRNA sequences found in the slush were closely related to planktonic bacteria from the water column beneath the ice. Conversely, during spring ablation, 50% of the sequences had > 97% identity with bacteria from the cryosphere (i.e. globally distributed glaciers, snow and ice) and may have originated from remote aerosol deposition. The transition winter to spring was characterized by consistent community changes switching from assemblages dominated by Betaproteobacteria, Verrucomicrobia and Bacteroidetes during snowpack growth to communities essentially dominated by the Bacteroidetes of classes Cytophagia and Sphingobacteria. This strong bacterial composition switch was associated with consistent increases in bacterial abundance and production, and decreasing bacterial diversity.This research was supported by grants PIRENA CGL2009-13318 and GOS-LAKES CGL2009-08523-E to E.O.C., and CONSOLIDER grant GRACCIE CSD2007-00067 from the Spanish Office of Science and Innovation (MICINN). J.C.A. benefits from a Juan de la Cierva postdoctoral fellow (MICINN).Peer reviewe

High Bacterial Diversity and Phylogenetic Novelty in Dark Euxinic Freshwaters Analyzed by 16S Tag Community Profiling

9 páginas, 4 figuras, 1 tablaMicrobial communities growing under extreme low redox conditions are present in anoxic and sulfide-rich (euxinic) environments such as karstic lakes and experience limitation of electron acceptors. The fine natural chemical gradients and the large diversity of organic and inorganic compounds accumulated in bottom waters are impossible to mimic under laboratory conditions, and only a few groups have been cultured. We investigated the bacterial composition in the oxic-anoxic interface and in the deep waters of three sulfurous lakes from the Lake Banyoles karstic area (NE Spain) through 16S rRNA gene tag sequencing and identified the closest GenBank counterpart. High diversity indices were found in most of the samples with >15 phyla/classes and >45 bacterial orders. A higher proportion of operational taxonomic units (OTUs) of the Bhighest novelty^ was found in the hypolimnia (38 % of total sequences) than in themetalimnia (17%),whereas the percentage of OTUs closer to cultured counterparts (i.e., 97 % identity in the 16S rRNA gene) was 6 to 21 %, respectively. Elusimicrobia, Chloroflexi, Fibrobacteres, and Spirochaetes were the taxa with the highest proportion of novel sequences. Interestingly, tag sequencing results comparison with metagenomics data available from the same dataset, showed a systematic underestimation of sulfuroxidizing Epsilonproteobacteria with the currently available 907R Buniversal^ primer. Overall, despite the limitation of electron acceptors, a highly diverse and novel assemblage was present in dark and euxinic hypolimnetic freshwaters, unveiling a hotspot of microbial diversity with a remarkable gap with cultured counterparts.This research was funded by grants GOS-LAKES CGL2009-08523-E and DARKNESS CGL2012-32747 to EOC and ARCANOX CGL2009-13318-C02-02 to CBM from the Spanish Office of Science (MINECO) and from the financial support by the Beyster Family Fund of the San Diego Foundation and the Life Technologies Foundation to the J. Craig Venter Institute.Peer reviewe

Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

14 páginas, 7 figuras, 3 tablas.Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO2 fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.This research was funded by Grants GOS-LAKES CGL2009-08523-E and DARKNESS CGL2012-32747 to EOC from the Spanish Office of Science (MINECO), from financial support by the Beyster Family Fund of the San Diego Foundation and the Life Technologies Foundation to the J Craig Venter Institute, and the NASA Astrobiology Institute to CLD.Peer reviewe

Speciation and ecological success in dimly lit waters: horizontal gene transfer in a green sulfur bacteria bloom unveiled by metagenomic assembly

11 páginas, 6 figuras.A natural planktonic bloom of a brown-pigmented photosynthetic green sulfur bacteria (GSB) from the disphotic zone of karstic Lake Banyoles (NE Spain) was studied as a natural enrichment culture from which a nearly complete genome was obtained after metagenomic assembly. We showed in situ a case where horizontal gene transfer (HGT) explained the ecological success of a natural population unveiling ecosystem-specific adaptations. The uncultured brown-pigmented GSB was 99.7% identical in the 16S rRNA gene sequence to its green-pigmented cultured counterpart Chlorobium luteolum DSM 273T. Several differences were detected for ferrous iron acquisition potential, ATP synthesis and gas vesicle formation, although the most striking trait was related to pigment biosynthesis strategy. Chl. luteolum DSM 273T synthesizes bacteriochlorophyll (BChl) c, whereas Chl. luteolum CIII incorporated by HGT a 18-kbp cluster with the genes needed for BChl e and specific carotenoids biosynthesis that provided ecophysiological advantages to successfully colonize the dimly lit waters. We also genomically characterized what we believe to be the first described GSB phage, which based on the metagenomic coverage was likely in an active state of lytic infection. Overall, we observed spread HGT and we unveiled clear evidence for virus-mediated HGT in a natural population of photosynthetic GSB.This research was funded by grant DARKNESS CGL2012- 32747 from the Spanish Office of Science (MINECO) to EOC and by the Global Ocean Sampling Project supported by the Beyster Family Foundation Fund of the San Diego Foundation and the Life Technology Foundation (to JCVI). Work on BChl e biosynthesis and the genomics of GSB in the laboratory of DAB was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-94ER20137.Peer reviewe