First Insights into the Viral Communities of the Deep-sea Anoxic Brines of the Red Sea

The deep-sea brines of the Red Sea include some of the most extreme and unique environments on Earth. They combine high salinities with increases in temperature, heavy metals, hydrostatic pressure, and anoxic conditions, creating unique settings for thriving populations of novel extremophiles. Despite a recent increase of studies focusing on these unusual biotopes, their viral communities remain unexplored. The current survey explores four metagenomic datasets obtained from different brine–seawater interface samples, focusing specifically on the diversity of their viral communities. Data analysis confirmed that the particle-attached viral communities present in the brine–seawater interfaces were diverse and generally dominated by Caudovirales, yet appearing distinct from sample to sample. With a level of caution, we report the unexpected finding of Phycodnaviridae, which infects algae and plants, and trace amounts of insect-infecting Iridoviridae. Results from Kebrit Deep revealed stratification in the viral communities present in the interface: the upper-interface was enriched with viruses associated with typical marine bacteria, while the lower-interface was enriched with haloviruses and halophages. These results provide first insights into the unexplored viral communities present in deep-sea brines of the Red Sea, representing one of the first steps for ongoing and future sampling efforts and studies

Exploring Deep-Sea Brines as Potential Terrestrial Analogues of Oceans in the Icy Moons of the Outer Solar System.

Several icy moons of the outer solar system have been receiving considerable attention and are currently seen as major targets for astrobiological research and the search for life beyond our planet. Despite the limited amount of data on the oceans of these moon, we expect them to be composed of brines with variable chemistry, some degree of hydrothermal input, and be under high pressure conditions. The combination of these different conditions significantly limits the number of extreme locations, which can be used as terrestrial analogues. Here we propose the use of deep-sea brines as potential terrestrial analogues to the oceans in the outer solar system. We provide an overview of what is currently known about the conditions on the icy moons of the outer solar system and their oceans as well as on deep-sea brines of the Red Sea and the Mediterranean and their microbiology. We also identify several threads of future research, which would be particularly useful in the context of future exploration of these extra-terrestrial oceans

Microbial assemblages in pressurized antarctic brine pockets (Tarn flat, northern Victoria land): A hotspot of biodiversity and activity

Two distinct pressurized hypersaline brine pockets (named TF4 and TF5), separated by a thin ice layer, were detected below an ice-sealed Antarctic lake. Prokaryotic (bacterial and archaeal) diversity, abundances (including virus-like particles) and metabolic profiles were investigated by an integrated approach, including traditional and new-generation methods. Although similar diversity indices were computed for both Bacteria and Archaea, distinct bacterial and archaeal assemblages were observed. Bacteroidetes and Gammaproteobacteria were more abundant in the shallowest brine pocket, TF4, and Deltaproteobacteria, mainly represented by versatile sulphate-reducing bacteria, dominated in the deepest, TF5. The detection of sulphate-reducing bacteria and methanogenic Archaea likely reflects the presence of a distinct synthrophic consortium in TF5. Surprisingly, members assigned to hyperthermophilic Crenarchaeota and Euryarchaeota were common to both brines, indicating that these cold habitats host the most thermally tolerant Archaea. The patterns of microbial communities were different, coherently with the observed microbiological diversity between TF4 and TF5 brines. Both the influence exerted by upward movement of saline brines from a sub-surface anoxic system and the possible occurrence of an ancient ice remnant from the Ross Ice Shelf were the likely main factors shaping the microbial communities

Prokaryotic and viral community of the sulfate‐rich crust from Peñahueca ephemeral lake, an astrobiology analogue

Peñahueca is an athalassohaline hypersaline inland ephemeral lake originated under semiarid conditions in the central Iberian Peninsula (Spain). Its chemical composition makes it extreme for microbial life as well as a terrestrial analogue of other planetary environments. To investigate the persistence of microbial life associated with sulfate‐rich crusts, we applied cultivation‐independent methods (optical and electron microscopy, 16S rRNA gene profiling and metagenomics) to describe the prokaryotic community and its associated viruses. The diversity for Bacteria was very low and was vastly dominated by endospore formers related to Pontibacillus marinus of the Firmicutes phylum. The archaeal assemblage was more diverse and included taxa related to those normally found in hypersaline environments. Several ‘metagenome assembled genomes’ were recovered, corresponding to new species of Pontibacillus, several species from the Halobacteria and one new member of the Nanohaloarchaeota. The viral assemblage, although composed of the morphotypes typical of high salt systems, showed little similarity to previously isolated/reconstructed halophages. Several putative prophages of Pontibacillus and haloarchaeal hosts were identified. Remarkably, the Peñahueca sulfate‐rich metagenome contained CRISPR‐associated proteins and repetitions which were over 10‐fold higher than in most hypersaline systems analysed so far.This research was supported by the Spanish Ministry of Economy projects CLG2015_66686-C3-1 (to RRM) CLG2015_66686-C3-3 (to JA), CGL2015-66455-R (to MAGC, MESM, JPRA), AYA2011-24803 and ESP2015-69540-R (to VP) which were also supported by the European Regional Development Fund and the MDM-2017-0737 Unidad de Excelencia “María de Maeztu”- Centro de Astrobiología (INTA-CSIC)

Comparative Genomics of the Genus Methanohalophilus, Including a Newly Isolated Strain From Kebrit Deep in the Red Sea

Halophilic methanogens play an important role in the carbon cycle in hypersaline environments, but are under-represented in culture collections. In this study, we describe a novel Methanohalophilus strain that was isolated from the sulfide-rich brine-seawater interface of Kebrit Deep in the Red Sea. Based on physiological and phylogenomic features, strain RSK, which is the first methanogenic archaeon to be isolated from a deep hypersaline anoxic brine lake of the Red Sea, represents a novel species of this genus. In order to compare the genetic traits underpinning the adaptations of this genus in diverse hypersaline environments, we sequenced the genome of strain RSK and compared it with genomes of previously isolated and well characterized species in this genus (Methanohalophilus mahii, Methanohalophilus halophilus, Methanohalophilus portucalensis, and Methanohalophilus euhalobius). These analyses revealed a highly conserved genomic core of greater than 93% of annotated genes (1490 genes) containing pathways for methylotrophic methanogenesis, osmoprotection through salt-out strategy, and oxidative stress response, among others. Despite the high degree of genomic conservation, species-specific differences in sulfur and glycogen metabolisms, viral resistance, amino acid, and peptide uptake machineries were also evident. Thus, while Methanohalophilus species are found in diverse extreme environments, each genotype also possesses adaptive traits that are likely relevant in their respective hypersaline habitats

MICROBIAL STRATIFICATION AND INFERRED MICROBIALLY CATALYZED PROCESSES ALONG A DEEP-SEA HYPERSALINE CHEMOCLINE

The Gulf of Mexico contains the world’s largest anoxic hypersaline seafloor basin, Orca Basin. The water contained in this 400 km2 bathymetric depression is roughly eight times as saline as the overlying seawater. The resulting density contrast prevents the 200 m deep brine layer from mixing with seawater, creating an interface that traps particles of organic matter falling through the water column. The concentrated organic matter at the interface is hypothesized to host a thriving bacterial community that has yet to be characterized. Here, I present the results of the first bacterial community analysis by high-throughput sequencing ever conducted on the interface and brine pool of Orca Basin. I discuss how the bacterial community changes along a 550 m vertical transect with regards to oxygen, salinity, and organic matter gradients. Finally, a comparison of the geochemical and bacterial composition of Orca Basin to brine pools in the Mediterranean and Red Seas reveals the uniqueness of Orca Basin in a global context. This research adds to our current knowledge of biodiversity in global hypersaline habitats and has implications for our understanding of sulfur and carbon cycling in extreme environmentsMaster of Scienc

Dead or alive: sediment DNA archives as tools for tracking aquatic evolution and adaptation

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress

Novel DNA ligases from the Red Sea brine pools: Cloning, expression, in silico characterization and comparative thermostability

Extreme physicochemical conditions such as high temperature, salinity, and the presence of heavy metal are characteristics of some of the Red Sea brine pools environment. We screened two Red Sea Brine pools (Atlantis II(AT-II), and Discovery Deeps (DD), and one interface layer (Kebrit Deep) to identify novel DNA ligases with potential extreme biochemical properties. Furthermore, we did an in silico comparative thermostability study by examining the stability role of proline and arginine residues at the loop conformations and exposed regions of ligase sequences from metagenomic assemblies of different extreme environments, including the Red Sea metagenomes. A sequence-based metagenomics approach was used to identify the putative DNA ligase sequences from the Red Sea brine pools and interface layer metagenomes downloaded from the NCBI database. 6, 148, 453 metagenomic reads were assembled using MEGAHIT, which generated 783,176 contigs. A concatenated HMM model built from raw HMM models of ATP and NAD+ ligases domains available from the Pfam database was used to scan predicted ORFs from contigs. A total of 18 ORFs were identified, and two of the ORFs, LigATL1 ATP type), from AT-II and LigKDU4 (NAD+ type) from KB, were selected for synthesis, phylogenetic study, and further preliminary characterizations. LigATL1 was cloned, expressed, and partially purified. Additionally, ligase sequences from psychrophilic, mesophilic, thermophilic, and hyperthermophilic environments were retrieved from the NCBI database for comparative thermostability study with some of the putative Red Sea ligase sequences. The retrieved 22 ligase sequences were divided into five different closest taxonomic groups. ConSurf and DisEMBL servers were used to analyze Proline (Pro) and Arginine (Arg) residues in the exposed/buried regions and the loop and hot loops regions of the putative ligases (retrieved + Red Sea), respectively. A putative LigATL1 showed a 38% identity to ATP-Dependent DNA ligase from Erysipelotrichaceae bacterium, while LigKDU4 has a 60% identity to NAD+ Dependent DNA ligase from Candidatus Marinimicrobia bacterium. The phylogenetic analysis suggests that LigATL1 belongs to the LigD(ATP type) family, while LigKDU4 is amongst the LigA family,(NAD+ type). LigATL1 has 100% confidence modeling using bound-adenylated nicked human DNA ligase as a template, and is superimposed with the highest similarity (Template modeling ™ score =1.0) to thermostable DNA ligase from S.solfataricus. LigKDU4 modeled with 100% confidence using bound-adenylated nicked E.coli DNA ligase, and also superimposed with the highest similarity(TM score= 1.0) to thermostable t2 filiform DNA ligase. In vitro, functional assay and biochemical characterization are still required to confirm both enzyme activity and thermostability. For the comparative thermostability analysis, many Ligase sequences from thermophilic or hyper thermophilic environments had higher Pro and Arg residues both at the exposed and the hot loops regions than those from other mesophilic and psychrophilic environments. The highest buried Pro and Arg residues were reported for ligase sequences from psychrophilic environments at almost all the groups. Two out of five putative ligase sequences selected for the thermophilic AT-II environment had more hot loops and less buried Pro and Arg residues than other pairs in their respective groups. In the case of LigKDU4(MLK), it has the highest hot loop and exposed Arg residues than its pairs in its group which is unusual when compared to Arg analysis in other groups. This comparative study can give an insight into improving the thermal stability of enzymes generally

Impact of a Major Inflow Event on the Composition and Distribution of Bacterioplankton Communities in the Baltic Sea

Major Baltic inflow (MBI) events carry highly saline water from the North Sea to the central Baltic Sea and thereby affect both its environmental conditions and its biota. While bacterioplankton communities in the Baltic Sea are strongly structured by salinity, how MBIs impact the composition and distribution of bacteria is unknown. The exceptional MBI in 2014, which brought saline and oxygenated water into the basins of the central Baltic Sea, enabled the linkage of microbiological investigations to hydrographic and modeling studies of this MBI. Using sequence data of 16S ribosomal RNA (rRNA) and 16S rRNA genes (rDNA), we analyzed bacterioplankton community composition in the inflowing water and in the uplifted former bottomwater at stations reached by the MBI. Bacterial diversity data were compared with respective data obtained from previous, non-inflow conditions. Changes in bacterial community composition following the 2014 MBI were mainly apparent at the genus level. A number of specific taxa were enriched in the inflowing water, with large changes in the rRNA/rDNA ratios indicating the different activity levels between of the water masses. The relative similarity of the bacterial communities in the inflowing and uplifted waters as well as the results from an inflow-simulating numerical model showed that the inflowing water did not originate directly from the North Sea but mostly from adjacent areas in the Baltic Sea. This suggested that the inflow event led to a series of shifts in Baltic Sea water masses among the Baltic Sea basins and a gradual mixing of the water bodies. Dramatic changes in the bacterial community composition occurred when the bottomwater inflow reached the anoxic, sulfidic deep basins, resulting in an uplifting of the formerly anoxic bacterial community, dominated by Epsilonproteobacteria. Our study of the impact of MBIs on bacterioplankton communities therefore highlights two relevant underlying mechanisms that impact the distribution and possibly also the activities of planktonic bacteria in the Baltic Sea: (1) the successive dilution of inflowing North Sea water with ambient waters and (2) the uplifting of former bottom-water communities to higher water strata.This work was funded by the Deutsche Forschungsgemeinschaft (DFG) (projects JU367/15-1, JU367/16-1 to KJ and LA1466/8- 1 to ML). DH was supported by the European Regional Development Fund and the Estonian Research Council Mobilitas Plus Top Researcher grant “MOBTT24.” UG was supported by the BMBF project “Hydrodynamic observations and simulations of munition in the sea,” a subproject of the collaborative project “Environmental monitoring for the delaboration of munitions in the sea” (Grant No. #03F0747C).This work was funded by the Deutsche Forschungsgemeinschaft (DFG) (projects JU367/15-1, JU367/16-1 to KJ and LA1466/8- 1 to ML). DH was supported by the European Regional Development Fund and the Estonian Research Council Mobilitas Plus Top Researcher grant “MOBTT24.” UG was supported by the BMBF project “Hydrodynamic observations and simulations of munition in the sea,” a subproject of the collaborative project “Environmental monitoring for the delaboration of munitions in the sea” (Grant No. #03F0747C)