Supplementary Document 2: Results

Osmolyte Production

Microorganisms living in hypersaline environments employ two general strategies for coping with osmotic stresses associated with low water activity. They can achieve osmotic stability by regulating concentrations of inorganic ions such as KCl (the ‘salt-in’ approach), or more commonly, they can produce and accumulate low-molecular-weight organic compounds such as amino acids and derivatives, alcohols, polyols (e.g., sugars, manitol, arabitol, glycerol) and their derivatives, betaines and thetines, and ectoines (the ‘salt-out’ approach), that protect enzymes and macromolecular structures from inactivation, inhibition, and denaturation under low water activity (Grant et al. 2004; Stan-Lotter and Fendrihan 2013). This strategy is widely observed among bacteria in hypersaline environments (Freeman et al. 2013). At the level of proteins, organisms must have adaptations as well, and several of these have been documented in other hypersaline habitats, such as lower concentrations of hydrophobic amino acids, and excesses of amino acids with acidic residues (Paul et al. 2008).

Differential expression of genes associated with adaptations to hypersalinity was evident in our halocline metatranscriptomes, and more abundant relative to the control sample (Figure 3). Consistent with overall low recovery of transcripts for L’Atalante, expression of putative osmotic protectants and ion transporters in the LLH sample was also low. The highest expression levels of putative osmolytes, transporters, or other genes indicative of osmotic stress responses was observed in sediments from athallasohaline Discovery, particularly Nha-type Na+/H+ antiporters, osmosensitive K+ channel histidine kinase (KdpD sensor kinase proteins involved in potassium transport and sensing turgor pressure), glutamate and glutamine synthetases, choline-glycine betaine transporters, choline dehydrogenase (possibly involved in betaine biosynthesis), and pyrroline-5-carboxylate reductase (can be involved in proline biosynthesis under osmotic stress (Perez-Arellano et al. 2010)). Higher expression in DLHvs. DUH sediments supports their involvement in adaptation to hypersalinity, and particularly to high MgCl2 concentrations. In the DLH sediments we detected transcripts of lysine 2,3-aminomutase, an enzyme that is known to be salt induced and essential for growth of methanogenicarchaea under hypersaline conditions (Pflüger et al. 2003). In Discovery and thallasohalineUrania basin halocline sediments, we recovered high expression of genes for biosynthesis of the amino acids glutamate and glutamine relative to the control sample, suggesting this is an important strategy for osmoprotection in these two DHABs. In addition, we observed relatively high levels of transcripts associated with choline dehydrogenase, Nha-type Na+/H+ antiporters, aspartate-semialdehyde dehydrogenase (can be involved in ectoine and hydroxyectoine biosynthesis as osmoprotectants (Stöveken et al. 2011)), and osmosensitive K+ channel histidine kinase. Lower expression of other genes with possible involvement in osmotic stress responses included ankyrin (involved in stress responses to environmental factors (Sakamoto et al. 2013), and aspartokinase (involved in ectoine and hydroxyectoine biosynthesis (Stöveken et al. 2011)).

Expresssion of Nha-type Na+/H+antiporters, as well as K+ transporters and K+/H+antiporters was observed in the Discovery and Urania halocline samples. This suggests adaptation of some microorganisms through the “salt-in” rather than “salt-out” strategy, consistent with what we observed previously in the water column halocline of Thetis DHAB (Pachiadaki et al. 2014) and with energetic constraints at high salinity described by Oren (2013). The presence of sodium-motive ferredoxin:NADoxidoreductase (Rnf) subunits in sediments from Urania and Discovery suggest some organisms may be capable of utilizing a sodium ion potential for energetic reactions (Biegel et al. 2011; Poehlein et al. 2012). Based solely on metatranscriptome data it is difficult to unambiguously determine that many of the transcripts for these putative osmolytes, transporters, and associated genes are involved exclusively in osmotic stress responses because most have multiple roles in cellular metabolism. Salicylate hydrolase was highly expressed the DLH sediments (annotated to an unknown Fungi, data not shown) and to a lesserextent in UMH. This enzyme is known in plants to play a role in plant responses to osmotic stress, however it can also be involved in responses to a wide range of environmental factors (Borsani et al. 2001). Experiments with cultured isolates from these habitats in the future could be used to test hypotheses about their roles.

Heavy Metals

Expression of genes associated with other heavy metals was also detected in our samples, including genes associated with molybdenum (Mb) metabolism in cores from Discovery lower halocline and Urania middle halocline, and to a much lesser extent in control and other DHAB samples (Supplementary Figure Mb). However, like Mb, some of these metals are required in small quantities for cell processes. The recovery of a broad selection of genes implicated in the detoxification of the heavy metals As and Hg predominantly in Discovery and Urania halocline sediments and not in L’Atalante or control sediments supports the hypothesis that DHAB metal concentrations are closely correlated with unique evaporate or brine sources for the different DHABs. Copper resistance gene transcripts were detected in all samples, including our control. Expression was the highest in Discovery lower halocline sediments, and we hypothesize that concentrations of copper may be relatively high in this DHAB. A similar pattern was observed for tellurium resistance transcripts.

Central Metabolism

Eukaryotic transcripts associated with central metabolism detected in UMH sediments exclusively included kynureinine 3-monooxygenase (involved in tryptophan metabolism and annotated to Penicillium), nucleolar proteins involved in 40S ribosome biogenesis (annotated mostly to Penicillium), a predicted endoplasmic reticulum membrane protein, and a RasGTPase-activating protein, both annotated to fungi. Expression of translation elongation factors 1, 2, and 3 (annotated to mixed fungi and protists) and DNA-directed RNA polymerase II (large subunit), and Ras related/Rac-GTP binding protein (Ras superfamily GTPases) were detected in both UMH and DLH sediments and annotated to fungi. Transcripts for a snoRNP protein involved in maturation of pre-18S rRNAof amoebae and fungi were detected in the DUH sediment sample.

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