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

Microbial 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 "highest novelty" was found in the hypolimnia (38 % of total sequences) than in the metalimnia (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 sulfur-oxidizing Epsilonproteobacteria with the currently available 907R "universal" 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.

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, 2240 m 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.

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

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.

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

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.

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

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.