Significance StatementMicrobial lipid membranes protect and isolate a cell from its environment and play a crucial role in cellular bioenergetics by regulating the flow of nutrients and metabolites to reaction centers within. We demonstrate that membrane lipids change as a function of energy flux using a well-studied archaeon that thrives in acidic hot springs and observe an increase in membrane packing as energy becomes more limited. These observations are consistent with chemostat experiments utilizing a low temperature, neutral pH, marine archaeon. This strategy to regulate membrane homeostasis is common across GDGT-producing lineages, demonstrating that diverse taxa adjust membrane composition in response to chronic energy stress.

Some of the most extreme marine habitats known are the Mediterranean deep hypersaline anoxic basins (DHABs; water depth ∼3500 m). Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB water-column haloclines and brines, the existence and activity of benthic DHAB protists have rarely been explored. Here, we report findings regarding protists and fungi recovered from sediments of three DHAB (Discovery, Urania, L’ Atalante) haloclines, and compare these to communities from sediments underlying normoxic waters of typical Mediterranean salinity. Halocline sediments, where the redoxcline impinges the seafloor, were studied from all three DHABs. Microscopic cell counts suggested that halocline sediments supported denser protist populations than those in adjacent control sediments. Pyrosequencing analysis based on ribosomal RNA detected eukaryotic ribotypes in the halocline sediments from each of the three DHABs, most of which were fungi. Sequences affiliated with Ustilaginomycotina Basidiomycota were the most abundant eukaryotic signatures detected. Benthic communities in these DHABs appeared to differ, as expected, due to differing brine chemistries. Microscopy indicated that only a low proportion of protists appeared to bear associated putative symbionts. In a considerable number of cases, when prokaryotes were associated with a protist, DAPI staining did not reveal presence of any nuclei, suggesting that at least some protists were carcasses inhabited by prokaryotic scavengers.

12 Significance Statement 13Microbial lipid membranes protect and isolate a cell from its environment while regulating 14 the flow of energy and nutrients to metabolic reaction centers within. We demonstrate that 15 membrane lipids change as a function of energy flux using a well-studied archaeon that 16 thrives in acidic hot springs and observe an increase in membrane packing as energy 17 becomes more limited. These observations are consistent with chemostat experiments 18 utilizing a low temperature, neutral pH, marine archaeon. This strategy appears to regulate 19 membrane homeostasis is common across GDGT-producing lineages, demonstrating that 20 diverse taxa adjust membrane composition in response to chronic energy stress. 21 Summary 22 23 Microorganisms regulate the composition of their membranes in response to environmental 24 cues. Many archaea maintain the fluidity and permeability of their membranes by adjusting 25 the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol 26 tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which 27 has been shown to help cells survive shifts in temperature and pH. However, the extent of 28 this cyclization also varies with growth phase and electron acceptor or donor limitation. 29 These observations indicate a relationship between energy metabolism and membrane 30 composition. Here we show that the average degree of GDGT cyclization increases with 31 Zhou et al. 2019 20190817 2 doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius 32 (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus 33 maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can 34 shift in response to electron donor flux and energy availability, independent of pH or 35 temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy 36 available to microbes, which encompasses fluctuations in temperature and pH, as well as 37 electron donor and acceptor availability. The ability of Archaea to adjust membrane 38 composition and packing may be an important strategy that enables survival during episodes 39 of energy stress. 40 41

Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT-making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses.

Oceanic protist grazing at mesopelagic and bathypelagic depths, and their subsequent effects on trophic links between eukaryotes and prokaryotes, are not well constrained. Recent studies show evidence of higher than expected grazing activity by protists down to mesopelagic depths. This study provides the first exploration of protist grazing in the bathypelagic North Atlantic Deep Water (NADW). Grazing was measured throughout the water column at three stations in the South Atlantic using fluorescently-labeled prey analogues. Grazing in the deep Antarctic Intermediate water (AAIW) and NADW at all three stations removed 3.79% ± 1.72% to 31.14% ± 8.24% of the standing prokaryote stock. These results imply that protist grazing may be a significant source of labile organic carbon at certain meso- and bathypelagic depths.