Cold adaptation of the Antarctic haloarchaea

Environmental Microbiology (2017) 19(6), 2210–2227

doi:10.1111/1462-2920.13705

Cold adaptation of the Antarctic haloarchaea Halohasta litchfieldiae and Halorubrum lacusprofundi

Timothy J. Williams,1† Yan Liao,1† Jun Ye,1,2 Rhiannon P. Kuchel,3 Anne Poljak,4 Mark J. Raftery4 and Ricardo Cavicchioli1* 1 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia. 2 Centre for Marine Bio-Innovation, The University of New South Wales, Sydney, New South Wales 2052, Australia. 3 Electron Microscopy Unit, The University of New South Wales, Sydney, New South Wales 2052, Australia. 4 Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, New South Wales 2052, Australia. Summary Halohasta litchfieldiae represents 44% and Halorubrum lacusprofundi 10% of the hypersaline, perennially cold ( 2208C) Deep Lake community in Antarctica. We used proteomics and microscopy to define physiological responses of these haloarchaea to growth at high (308C) and low (10 and 48C) temperatures. The proteomic data indicate that both species responded to low temperature by modifying their cell envelope including protein N-glycosylation, maintaining osmotic balance and translation initiation, and modifying RNA turnover and tRNA modification. Distinctions between the two species included DNA protection and repair strategies (e.g. roles of UspA and Rad50), and metabolism of glycerol and pyruvate. For Hrr. lacusprofundi, low temperature led to the formation of polyhydroxyalkanoate-like granules, with granule formation occurring by an unknown mechanism. Hrr. lacusprofundi also formed biofilms and synthesized high levels of Hsp20 chaperones. Hht. litchfieldiae was characterized by an active CRISPR system, and elevated levels of the core gene expression machinery, which contrasted markedly to Received 30 October, 2016; revised 17 January, 2017; accepted 8 February, 2017. *For correspondence. E-mail: r.cavicchioli@unsw. edu.au; Tel. 161 2 9385 3516; Fax 161 2 9385 2742. †These authors contributed equally to this work. C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd V

the decreased levels of Hrr. lacusprofundi. These findings greatly expand the understanding of cellular mechanisms of cold adaptation in psychrophilic archaea, and provide insight into how Hht. litchfieldiae gains dominance in Deep Lake.

Introduction Cold environments dominate the Earth’s biosphere and the indigenous microorganisms perform critical processes that make major contributions to global biogeochemical cycles (Margesin and Miteva, 2011; Cavicchioli, 2015). In the cold biosphere, archaea are prevalent and are represented by diverse lineages (Cavicchioli, 2006). However, as is typical for environmental microorganisms, few psychrophilic archaea have been isolated and even fewer have been studied to determine molecular mechanisms of cold adaptation (Cavicchioli, 2006). The best studied is Methanococcoides burtonii, a methylotrophic methanogen isolated from Ace Lake, Antarctica (Cavicchioli, 2006; Najnin et al., 2016; Taha et al., 2016 and references therein). While M. burtonii has served well as a model psychrophilic archaeon, it must be grown anaerobically, has not been cultivated on solid media, and a genetic manipulation system has not been developed for it. Other archaea from naturally cold environments that have been studied include Methanogenium frigidum, SM1 Euryarchaeon, Cenarchaeum symbiosum and Halorubrum lacusprofundi (reviewed in Cavicchioli, 2006), Methanosarcina SMA-21 (Morozova and Wagner, 2007) and Methanolobus psychrophilus (Chen et al., 2012; Chen et al., 2015). Approximately 10 km from Ace Lake where M. burtonii was isolated is Deep Lake. It is a 36 m deep, hypersaline system that has the distinction of being the lowest accessible point on the Antarctic continent, the least productive lake system known, the coldest aquatic environment known to support life, and the source of the first formally described member of the Archaea from a cold environment (Hrr. lacusprofundi) (reviewed in Cavicchioli et al., 2011; Cavicchioli, 2015). The lake is dominated by a low complexity, hierarchically structured community of haloarchaea that differs markedly to the species typical of warmer environments (DeMaere et al., 2013). Halohasta litchfieldiae is the most abundant species representing 44%, and Hrr.

Cold adaptation of Antarctic haloarchaea 2211 lacusprofundi is the third most abundant species representing 10% (DeMaere et al., 2013). Genomic-, metagenomic- and metaproteomic-led studies have identified ecophysiological traits that describe trophic distinctions of the dominant species, and host-virus interactions associated with gene transfer and host evasion, defense and adaptation (Williams et al., 2014; Tschitschko et al., 2015; Tschitschko et al., 2016). Laboratory cultivation of Hht. litchfieldiae and Hrr. lacusprofundi enables fastest rates of growth for both organisms at 308C, with growth continuing until temperatures reach 42–458C (Franzmann et al., 1988; Mou et al., 2012). Growing the strains in the laboratory below about 48C is difficult, with Hrr. lacusprofundi exhibiting minimal growth over 30 days at 0 or 218C (Reid et al., 2006). However, in Deep Lake where these species dominate, water temperatures drop to 2208C and remain subzero at depth throughout the year (e.g. 29 to 2178C below 20 m), with only surface waters in summer reaching temperatures up to 10 2 128C (Campbell, 1978; Barker, 1981; Ferris and Burton, 1988). The fact that microorganisms from cold environments have the capacity to grow faster at temperatures that exceed their natural environment has led to the misconception that they are not adapted to the cold and are not ‘true’ psychrophiles (discussed in Feller and Gerday, 2003; Cavicchioli, 2006; Cavicchioli, 2016). As Hht. litchfieldiae and Hrr. lacusprofundi represent 56% of the entire lake population in Deep Lake, they illustrate well why laboratory measured growth rates provide a poor estimate of ecological competitiveness. Quantitative proteomic and transcriptomic studies of M. burtonii have been successfully used to shed light on the molecular responses of cells grown at different temperatures, including studies that illustrate that temperatures producing fast growth rates can cause heat stress (e.g. Campanaro et al., 2011; Williams et al., 2011). Recently for Hrr. lacusprofundi, a system for genetic manipulation was developed (Liao et al., 2016a), and quantitative proteomics performed using isobaric tags for quantification (iTRAQ) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize proteins involved in biofilm formation (Liao et al., 2016b). To advance the understanding of growth temperaturedependent responses of Antarctic haloarchaea, Hht. litchfieldiae and Hrr. lacusprofundi were grown at 30, 10 and 48C and iTRAQ proteomics performed to identify and quantify proteins. Microscopy was also performed to identify morphological features characteristic of growth temperature, with proteomics used to identify the proteins and cellular processes that are likely to be involved. The analyses allowed conclusions to be drawn about responses that are common to the two Antarctic haloarchaea, and may be broadly representative of cold adaptation in psychrophilic archaea, as well as specific traits that likely

contribute to the numerical dominance of Hht. litchfieldiae over Hrr. lacusprofundi in Deep Lake. Results and discussion Proteomics overview Proteomics was performed on Hht. litchfieldiae and Hrr. lacusprofundi grown to mid-logarithmic phase at 30, 10 and 48C, with proteins analysed from both the whole-cell fraction and extracellular (supernatant) fraction (Supporting Information Tables S1 and S2), using approaches previously developed for Hrr. lacusprofundi (Liao et al., 2016b) and M. burtonii (Williams et al., 2010). Proteins from Hht. litchfieldiae are denoted halTADL, and Hlac for Hrr. lacusprofundi. The extracellular fraction was used primarily to assess changes in secreted cell envelope proteins. The 8plex iTRAQ labelling was used to determine abundance differences between cells grown at 4 vs. 308C and 10 vs. 308C. The trends for these two comparisons were similar, with more pronounced abundance differences between 4 and 308C than 10 and 308C (Supporting Information Tables S1 and S2). As a result, the proteomic descriptions below are mainly described in terms of differences at low (4 or 108C) vs. high (308C) temperature. As a proportion of total protein-coding genes, 39% (1341 proteins) and 43% (1559 proteins) proteome coverage was obtained for Hht. litchfieldiae and Hrr. lacusprofundi, respectively. Growth, morphology, and cell envelope proteins At the temperatures used for cultivation (30, 10, and 48C), Hht. litchfieldiae tended to grow faster and reached a higher final OD600 than Hrr. lacusprofundi, with the difference being most noticeable at 48C (Fig. 1). Hrr. lacusprofundi cultures formed biofilms at mid-logarithmic and stationary phases at 48C and 108C (Fig. 2A–C), with biofilms more abundant and appearing earlier in the growth phase at 48C than 108C. In contrast, Hht. litchfieldiae did not form biofilms under the growth conditions tested (Fig. 3A–C). Scanning electron microscopy revealed that Hrr. lacusprofundi cells were pleomorphic, with rod and coccus shapes present at the three growth temperatures (Fig. 2D– F), consistent with previous descriptions of the organism (Franzmann et al., 1988; Liao et al., 2016b). A higher proportion of cells were rod-shaped at low temperature, and particularly 48C-grown cells exhibited clumping with thread-like structures connecting cells (Fig. 2F). In addition, cell surface texture of Hrr. lacusprofundi changed from smooth (308C; Fig. 2D) to wrinkled at low growth temperatures (48C; Fig. 2F), and multiple short extracellular threads were present on some cells at low temperature (Fig. 2F); these structures were similar in appearance to those previously reported for Hrr. lacusprofundi biofilms that formed during growth in media that lacked ammonium

C 2017 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 19, 2210–2227 V

2212 T. J. Williams et al.

Fig. 1. Growth curves for the Antarctic haloarchaea. Hht. litchfieldiae (䊏) and Hrr. lacusprofundi (ⵧ) grown at 308C (A), 108C (B) and 48C (C). For microscopy and proteomics, cells were harvested at mid-logarithmic phase (M). Hht. litchfieldiae reached stationary phase at OD600 0.5 in 14 d at 308C, OD600 0.35 in 140 d at 108C, and OD600 0.35 in 250 d at 48C. Hrr. lacusprofundi reached stationary phase at OD600 0.4 in 14 d at 308C, OD600 0.3 in 165 d at 108C, and OD600 0.2 in 280 d at 48C.

(Liao et al., 2016b). Hht. litchfieldiae cells were predominantly rod-shaped at 308C (Fig. 3D), consistent with the original description of the organism (Mou et al., 2012). Hht. litchfieldiae cell length did not simply correlate with growth temperature with length appearing to be shorter at 108C compared to 308C or 48C (Fig. 3D–F). The temperature-dependent morphological and biofilm characteristics (Figs 2 and 3) were reflected in the proteomic data for cell envelope proteins (Supporting Information Tables S1 and S2). The protective outer surface layer (Slayer) of haloarchaea is composed of a glycosylated protein that forms a highly porous, paracrystalline lattice (Albers and Meyer, 2011). Hht. litchfieldiae encodes one S-layer protein (halTADL_1043), and Hrr. lacusprofundi encodes two (Hlac_0412, Hlac_2976) (Tschitschko et al., 2015). At low temperature, the abundance of the halTADL_1043 protein decreased in the extracellular fraction but not in the whole cell fraction, whereas the abundance of the Hlac_0412 protein decreased in the whole cell fraction but not the extracellular fraction. As S-layer proteins of haloarchaea can be released into the medium (Boot and €nig et al., 2007), the proteome data for Pouwels, 1996; Ko Hht. litchfieldiae may indicate that at low temperature, Slayer coverage of the cell did not change but less protein was released into the extracellular medium. In contrast for Hrr. lacusprofundi, S-layer production appeared to decrease at low temperature with the amount released from the cell remaining constant (Supporting Information Tables S1 and S2); these characteristics may relate to the shift to a biofilm phenotype in Hrr. lacusprofundi. Abundance differences between the whole cell fraction and extracellular fraction may also relate to the mode of

attachment of the S-layer glycoproteins to the cell membrane. In Haloferax volcanii, the S-layer proteins can be directly anchored to the membrane via their C-terminal transmembrane domain, or attach to the membrane via a lipid anchor that is formed during extracytoplasmic cleavage of the protein (Haft et al., 2012; Kandiba et al., 2013; Kandiba and Eichler, 2014). The cleaved form may arise during maturation and be shed into the medium without the cell membrane being compromised (Eichler, 2001; Kandiba et al., 2013). While the environmental conditions that regulate the relative abundance of the two forms have not been determined (Kandiba et al., 2013), our data suggest growth temperature may play a role in regulating the proportion of each form. N-glycosylation of S-layer proteins is required for assembly and function of the S-layer (Eichler et al., 2013; Jarrell et al., 2014), and growth conditions can regulate the pattern of glycosylation (Guan et al., 2012; Kaminski et al., 2013). For both species, a glycosyltransferase (halTADL_1783; Hlac_0164) that is homologous to a Hfx. volcanii protein (HVO_1613; dolichyl-phosphate hexose transferase) involved in the assembly of the glycan used in S-layer N-glycosylation (Kaminski et al., 2010), had higher abundance at low temperature. However, an NADdependent isomerase which is a homolog of an Nglycosylation enzyme (Agl12) involved in synthesis of a tetrasaccharide attached to the S-layer glycoprotein of Hfx. volcanii during ‘low-salt’ (1.75 M vs. 3.4 M) growth (Kaminski et al., 2013), had higher abundance at low temperature for Hrr. lacusprofundi (Hlac_1891) but lower abundance for Hht. litchfieldiae (halTADL_3057). Similarly, homologs of the nucleotidyltransferase (Agl11) that is also involved in synthesis of the ‘low-salt’ glycan in Hfx. volcanii (Kaminski


Cold adaptation of Antarctic haloarchaea 2213

Fig. 2. Growth temperature-dependent morphology of Hrr. lacusprofundi. Cells grown at 308C (A, D), 108C (B, E) and 48C (C, F) to mid-logarithmic phase (see Fig. 1) in flasks to observe if biofilms formed (A–C) and examined by SEM to assess cell morphology (D–F). D,E,F: scale bar, 10 mm. Images for cells that formed biofilms at 108C and 48C were recorded at two different magnifications, with magnified sections of E and F shown as insets with a scale bar of 1 mm.

et al., 2013) had higher (halTADL_3353; Hlac_1080) or lower (Hlac_2874) abundance at low temperature, as did several glycosyltransferases, with Hlac_0548 higher and halTADL_2565 and halTADL_3361 lower at low temperature. The proteomic data indicate that S-layer glycosylation

is regulated by growth temperature in specific ways for Hht. litchfieldiae and Hrr. lacusprofundi. Sulfation of S-layer glycans in Hrr. lacusprofundi may also be growth temperature regulated. A UDP-sulfoquinovose synthase (SqdB; Hlac_1075) that synthesizes the sulfated


2214 T. J. Williams et al.

Fig. 3. Growth temperature-dependent morphology of Hht. litchfieldiae. Cells grown at 308C (A, D), 108C (B, E) and 48C (C, F) to mid-logarithmic phase (see Fig. 1) in flasks to observe if biofilms formed (A–C) and examined by SEM to assess cell morphology (D–F). D,E,F: scale bar, 10 mm.

sugar sulfoquinovose, and phosphoadenosine phosphosulfate reductase (Hlac_1106) which provides the activated sulfate donor for sulfation (Honke and Taniguchi, 2002), were both less abundant at low temperature. When incorporated

into S-layer glycoproteins, sulfoquinovose increases the negative charge density of the S-layer (Mengele and Sumper, 1992), and a Sulfolobus acidocaldarius sqdB deletion mutant had impaired growth in high salt concentrations (Meyer et al.,


Cold adaptation of Antarctic haloarchaea 2215 2011). One hypothesis is that more negatively charged glycans create a hydrated shell that enhances stabilization of the S-layer (Mengele and Sumper, 1992), implying that decreased sulfation of the Hrr. lacusprofundi S-layer at low temperature might reduce negative charge density thereby creating a more flexible S-layer. Both species (especially Hrr. lacusprofundi) were also characterized by the differential abundance of numerous other secreted proteins (Supporting Information Tables S1 and S2). Based on the typical characteristics of archaeal secreted proteins (Szabo and Pohlschroder, 2012), these were likely to have been attached to the S-layer or anchored to the cell membrane. Decreased at low temperature were halTADL_1042 which contains a CARDB (cell adhesion related domain found in bacteria)/Ig-like fold domain, and Hlac_1583 which is homologous to a bacterial extracytoplasmic endopeptidase (Garnier et al., 1985; Hourdou et al., 1993). Conversely, showing increased abundance were Hlac_0123 and Hlac_0125, which contain von Willebrand factor type A domains involved in proteinprotein interactions (Colombatti et al., 1993); the putative DNA binding proteins halTADL_0044 (Tschitschko et al., 2016); the archaellum (archaeal flagellum) protein halTADL_1810; and Hlac_1867, which is a nuclease lipoprotein inferred to function in extracytoplasmic DNA metabolism (Chimileski et al., 2014). Post-translational modification of the S-layer via glycosylation and proteolysis, and involvement of Hlac_1867 in DNA metabolism, were previously inferred using proteomics as fulfilling roles in biofilm formation in Hrr. lacusprofundi during growth in media lacking ammonium (Liao et al., 2016b). The increased abundance at low temperature of tubulinlike CetZ proteins (CetZ1, Hlac_1892; CetZ2, Hlac_0198) may reflect the temperature-dependent changes observed in cell morphology (Fig. 2). These proteins have been shown to control cell shape in Hfx. volcanii (Duggin et al., 2015), and may therefore be involved in increasing the proportion of rod-shaped Hrr. lacusprofundi cells at low temperature (Fig. 2E and F). Overall these data point strongly to specific changes in cell surface and cell shape proteins, and modification to both cell surface proteins and their sugars, in adaptation of the Antarctic haloarchaea to growth at low temperature. This reinforces the view generated from studies of the Antarctic methanogen M. burtonii that remodelling of the cell envelope is central to enabling low temperature growth of cold-adapted archaea (Williams et al., 2010; Williams et al., 2011). Cell membrane and osmotic balance Proteome abundance changes indicative of alterations to membrane lipids included increased levels of hydroxymethylglutaryl-CoA (HMG-CoA) synthase

(halTADL_0750; Hlac_2313), which is involved in the synthesis of HMG-CoA that is required for isoprenoid chains of the core lipids synthesized via the mevalonate pathway (Kates, 1993), and lower levels of geranylgeranyl reductase (halTADL_1048, halTADL_3215; Hlac_0926), which is involved in selective saturation of newly formed lipids (Nichols et al., 2004). Elevated levels of unsaturated lipids have been identified in Hrr. lacusprofundi and M. burtonii at low growth temperatures (Nichols et al., 2004; Gibson et al., 2005). The proteomic and lipid data are consistent with a view that increasing the proportion of unsaturated isoprenoid lipids in psychrophilic archaea helps maintain membrane fluidity at low temperature (Nichols et al., 2004; Gibson et al., 2005; Cavicchioli, 2006). Halophiles need to maintain a cytoplasm that is isoosmotic with the surrounding environment (Oren, 2008), and to achieve this haloarchaea tend to use a ‘salt-in’ strategy involving the importation of K1 and Cl2 ions and export of Na1 ions (Oren, 2002). Low temperature growth increased the abundance of small-conductance mechanosensitive channels (halTADL_0380; Hlac_1441) that release osmolytes to prevent cell lysis (Wilson et al., 2013), and NhaP Na1/H1 antiporters (halTADL_2587, Hlac_0112, Hlac_1889) that produce Na1 efflux (Waditee et al., 2002). These increases in abundance may compensate for reduced rates of enzymatic activity at low temperature to ensure osmotic balance is maintained. Na1 ions are cotransported during the uptake of organic solutes by Na1dependent symporters, and for Hrr. lacusprofundi these had lower abundance at 48C (Hlac_0216, Hlac_1637). In this context it is noteworthy that fully unsaturated phytanyl chains can result in increased membrane permeability (Dannenmuller et al., 2000; Dawson et al., 2012), rendering cells more ‘leaky’, and necessitating the increased capacity for excluding Na1. Increased permeability to external cations would also likely increase the amount of ammonium ions entering the cell via diffusion. This could account for the decreased abundance of ammonium transporters at low temperature (halTADL_1826; Hlac_2623) if a higher proportion of ammonium enters the cell by passive diffusion. The proteomic data describe a combination of mechanisms to enable both cold adaptation and ‘salt-in’ strategy to be accommodated by the psychrophilic haloarchaea. Nutrient uptake and general metabolism The substrate-binding extracytoplasmic lipoprotein components of ATP-binding cassette (ABC) transporters of Hht. litchfieldiae and Hrr. lacusprofundi exhibited markedly different abundances in response to growth at low temperature that are consistent with different substrate preferences. Hht. litchfieldiae exhibited increased abundances of lipoproteins that target carbohydrates


2216 T. J. Williams et al. (halTADL_1911, halTADL_1095), whereas Hrr. lacusprofundi had increased abundances of lipoproteins that target a more diverse range of nutrients: carbohydrates (Hlac_2520), branched-chain amino acids (BCAAs) (Hlac_1092, Hlac_1149), oligopeptides (Hlac_0674), phosphate (Hlac_0378), and various metal cations (especially iron) (Hlac_2057, Hlac_0162, Hlac_0541, Hlac_0537, Hlac_0880). The data are consistent with the highly saccharolytic metabolism inferred for Hht. litchfieldiae, compared to a more versatile, less specialized metabolism for Hrr. lacusprofundi (Williams et al., 2014; Tschitschko et al., 2016). It is possible that an increase in substratebinding lipoproteins compensates for reduced substrate uptake arising from decreased binding affinity for the substrate and/or decreased ATP hydrolysis. Increased demand for substrates in cold environments has been attributed to decreased membrane lipid fluidity and the kinetic effect of low temperature on transporter function (Russell, 2009). For some Hrr. lacusprofundi ABC transporters, the cognate ATPase component had lower abundance (Hlac_0377, Hlac_2057), which might indicate a shift to substrate recruitment while minimizing bioenergetic investment (ATP hydrolysis). Rather than an effect on transport efficiency, lower abundance of a nucleosidebinding ABC transporter lipoprotein (Hlac_1416) may indicate a decreased demand for nucleosides linked to decreased DNA synthesis and cell replication. Both species also had higher levels of tripartite ATP-independent periplasmic (TRAP) transporters at low temperature, which use a proton motive force rather than ATP to drive the translocation of substrates (such as carboxylic acids) into the cytoplasm (Forward et al., 1997). The two species also exhibited different responses to low temperature for tricarboxylic acid (TCA) cycle enzymes. For Hht. litchfieldiae, acetyl-CoA synthetase (halTADL_1017) and TCA cycle enzymes up to the steps leading to fumarate, were more abundant at low temperature. The higher abundance of malic enzyme (halTADL_0683) and lower abundance of PEP carboxylase (halTADL_0401) at low temperature suggests that oxaloacetate and malate can be sourced through the carboxylation of pyruvate. Hrr. lacusprofundi TCA cycle enzymes had lower abundance, despite the higher abundance of acetylCoA synthetase (Hlac_0990). This suggests that a high proportion of the acetyl-CoA pool is drawn off for processes outside of the TCA cycle, such as fatty acid synthesis via acetoacetyl-CoA, hydroxyalkanoate synthesis via malonyl-CoA, and isoleucine synthesis via citramalate. Some of these changes for Hrr. lacusprofundi may also be linked to biofilm formation as acetyl-CoA production leading to the synthesis of specific carbohydrates was previously reported to contribute to Hrr. lacusprofundi biofilms (Liao et al., 2016b).

Glycerol metabolism As an osmolyte produced in large amounts in hypersaline environments by the primary producer Dunaliella, glycerol is an important nutrient for many haloarchaea (Oren, 1999; Williams et al., 2017) and is inferred to be a major carbon and energy source for Hht. litchfieldiae and to a lesser extent Hrr. lacusprofundi in Deep Lake (DeMaere et al., 2013; Williams et al., 2014; Tschitschko et al., 2016). For the proteomes of both species, glycerol kinase (GlpK) homologs were among the highest differentially abundant proteins at low temperature (halTADL_0681, halTADL_2249; Hlac_1122), as were the Hht. litchfieldiae glycerol-3-phosphate dehydrogenase (GlpABC) (halTADL_2248/2247/2246) and dihydroxyacetone (DHA) kinase (halTADL_2260/2259) enzymes. DHA kinase is dependent on a cytosolic phosphoenolpyruvate (PEP) dependent phosphotransferase system (Falb et al., 2008), and PEP synthase (halTADL_1210) had higher abundance at low temperature. As glycerol was not supplied in the growth medium, the elevated levels of glycerol catabolism enzymes could reflect: (1) regulatory mechanisms enabling responses to changes in environmental temperature but a ‘hard wiring’ of carbon metabolism (especially Hht. litchfieldiae) towards the utilization of the perennial availability of this substrate; (2) the glycerol-1-phosphate moiety of membrane lipids (e.g. from lysed cells) providing a potential source of glycerol for catabolism. This latter possibility is speculative because the degradation and turnover of haloarchaeal glycerophospholipids requires cleavage of ether bonds, and a mechanism to achieve this has not yet been described in archaea.

Fatty acid and propionyl-CoA metabolism Fatty acid metabolism in Hrr. lacusprofundi appears to be enhanced at low temperature through the increased abundance of acetyl-CoA acetyltransferase (Hlac_1850), acylactivating proteins (Hlac_0820, Hlac_2549), acyl-CoA dehydrogenase (Hlac_1141), enoyl-CoA hydratase (Hlac_1117), and 3-hydroxyacyl-CoA dehydrogenase (Hlac_1743). The latter three enzymes had higher abundance in Halobacterium salinarum NRC-1 under low-salt conditions (Leuko et al., 2009). Although archaeal membrane lipids do not contain fatty acids, certain fatty acids are constituents of membrane proteins (Falb et al., 2008). Thus, it has been proposed that these enzymes function in fatty acid synthesis (with intermediates activated with CoA rather than acyl-carrier protein), with the increased synthesis of long-chain fatty acids possibly stabilizing membranebound energy-transducing systems (e.g. cytochrome c oxidase) (Dibrova et al., 2014) and/or contributing to pez-Garcıa et al., 2015). increased membrane flexibility (Lo



Fig. 4. Transmission electron micrographs of PHA-like granules in Hrr. lacusprofundi. Images of logarithmic-phase Hrr. lacusprofundi cells grown at 48C. Scale bar, 1 or 2 mm.

At low temperature Hrr. lacusprofundi also showed higher abundance of proteins associated with propionylCoA synthesis, including acetyl-CoA carboxylase carboxyltransferase (Hlac_2454), biotin carboxylase (Hlac_2540), acyl-CoA synthetase (Hlac_1306), enoyl-CoA hydratase (Hlac_1117), and acrolyl-CoA reductase (Hlac_1815) (Han et al., 2013). Propionyl-CoA is a key precursor of polyhydroxyalkanoate (PHA) storage product (Han et al., 2013). Candidate genes for a pathway that leads from propionylCoA to hydroxyacyl-CoA are encoded in the Hrr. lacusprofundi genome, including a short-chain dehydrogenase/ reductase (SDR) superfamily protein (Hlac_1094), which also had higher abundance at low temperature, and might serve as acetoacetyl-CoA reductase. PHA-like granules were detected in Hrr. lacusprofundi using Nile Blue A staining, with cells grown at 308C showing very limited fluorescence, but strong fluorescence observed for cells grown at low temperature (Supporting Information Fig. S1). The presence of PHA-like granules in Hrr. lacusprofundi was confirmed by examining cells grown at low temperature using transmission electron microscopy (Fig. 4). PHA granules consist of an amorphous polymer

core enclosed by a proteinaceous surface layer that includes PHA synthase, phasins, and depolymerizing enzymes (Grage et al., 2009). Unlike Hht. litchfieldiae, where genes for PHA synthesis have been identified (Williams et al., 2014), the genome of Hrr. lacusprofundi lacks identifiable genes for any of these proteins; thus, a process for PHA granule formation in Hrr. lacusprofundi remains unknown. One protein that might be involved in hydroxyacyl-CoA polymerization to PHA is a multi-domain protein (Hlac_1732) that has 47% sequence identity in its C-terminal region with the equivalent C-terminal region of PHA synthase PhaC subunit, and had higher abundance at low temperature (Supporting Information Fig. S2). Homologs of this protein are found only in Halorubrum spp., and contain a central PASTA (penicillin and Ser/Thr kinase associated) domain involved in ligand binding (e.g. stem peptides of unlinked peptidoglycan) (Yeats et al., 2002; Jones and Dyson, 2006). Low temperature also produced higher abundances of BCAA ABC transporter proteins (see Nutrient uptake and general metabolism) and enzymes involved in BCAA synthesis (Hlac_0588, Hlac_0870, Hlac_0873/0874/0875,


2218 T. J. Williams et al. Hlac_0353). An increased production of PHA might be linked to the demand for BCAAs, since propionyl-CoA can also be generated by degradation of BCAAs or their imme€chel and Pieper, 1992). diate oxoacid precursors (Steinbu PHA is synthesized as a carbon and energy reserve and mobilized when needed via b-oxidation; this process requires the same enzymes involved in fatty acid synthesis to operate in the reverse direction (Liu et al., 2016). The higher abundance of both hydroxyacyl-CoA synthesis (via propionyl-CoA) and b-oxidation enzymes in Hrr. lacusprofundi is consistent with concurrent operation of PHA accumulation and degradation pathways, which has been observed in PHA-producing bacteria, and associated with intracellular levels of acetyl-CoA/CoA and NADH/NAD1 (Ren et al., 2009). Maintaining genome integrity and CRISPR defense The proteomic data are supportive of an increased requirement for mechanisms to maintain genomic integrity and effect DNA repair at low temperature. DNA repair and recombination protein RadA (halTADL_2135; Hlac_2624), which catalyzes strand invasion and exchange during homologous recombination (HR) and enables the rescue of stalled replication forks and recombinational repair (Woods and Dyall-Smith, 1997; McCready et al., 2005; Boubriak et al., 2008), had higher abundance at low temperature. Other DNA repair proteins included HerA helicase (halTADL_0836; Hlac_1979) which participates in DNA-end resection in preparation for HR repair of doublestranded breaks (DSB) (Blackwood et al., 2012), and methylated-DNA-protein cysteine methyltransferase (halTADL_0579; Hlac_1270) which participates in the repair of alkylated DNA (Olsson and Lindahl, 1980). Specific responses of the two species were also evident with Rad50, a protein involved in the DSB repair using HR (Delmas et al., 2009), having lower abundance at low temperature for halTADL_0967 but higher for Hlac_2789. In Hfx. volcanii, Rad50 initially restrains HR-mediated DSB repair to allow for alternate repair mechanisms that join DNA ends. DSB repair by HR is potentially hazardous to polyploid haloarchaeal cells since each DNA end has multiple potential partners and Rad50 is thought to be needed for recovery from DNA damage as any outstanding DNA ends are ultimately repaired by HR (Delmas et al., 2009). Our data for Rad50 suggest that each species may adopt a different approach to this trade-off, with Hht. litchfieldiae enabling faster but potentially more error-prone repair of DSB and Hrr. lacusprofundi providing more opportunity for non-HR DNA repair processes. Differences in the repair strategies for the two species were also reflected by higher abundances at low temperature for: 1) replication protein A single-stranded DNA-binding complex components (halTADL_3433/_3434) which are proposed to form during

DNA-damage repair in haloarchaea (McCready et al., 2005); 2) apurinic/apyrimidinic (AP) endonuclease (halTADL_2345) which carries out excision of the DNA phosphodiester backbone at AP sites in preparation for DNA polymerase repair synthesis (Garcin et al., 2008); 3) a DNA integrity scanning protein (Hlac_1442) that checks DNA for lesions (Bejerano-Sagie et al., 2006). Nine Hrr. lacusprofundi UspA and one Dps mini-ferritin (Hlac_0536) protein, and two Hht. litchfieldiae UspA proteins, all had higher abundance at low temperature. Increased UspA abundance has been linked to a response to DNA damage in a halophilic methanogen (Shih and Lai, 2010), and oxidative damage and growth-arrest in E. coli € m and Neidhardt, 1994; Kvint et al., 2003; Nachin (Nystro et al., 2005). Likewise, Dsp mini-ferritins may protect DNA from oxidative damage by depleting ferrous iron and hydrogen peroxide (Arnold and Barton, 2013), or fulfil roles as non-specific DNA-binding proteins that can protect DNA during growth cessation (Almiron et al., 1992). For both Antarctic haloarchaea, the reduced abundance at low temperature of components of the NADH oxidoreductase complex, ATP synthase subunits, and inorganic pyrophosphatase, are indicative of a lower energy metabolism and decreased potential for the formation of reactive oxygen species (ROS) via the electron transport chain. Hht. litchfieldiae superoxide dismutase (halTADL_2687) and Hrr. lacusprofundi catalase/peroxidase (Hlac_1548) were also much less abundant at low temperature. These data indicate that, despite the increased solubility of ROS at low temperature, it is unlikely that oxidative stress caused by ROS was the main cause of DNA damage at low temperature. However, replication fork arrests and fork damage might be exacerbated at low temperature due to DNA secondary structures and DNA-protein complexes becoming more stable (Sinha et al., 2013). Because stalled replication forks can be repaired by DSB and HR allowing replication to proceed (Kuzminov, 1995; Cox et al., 2000), the data suggest that the increase in DNA repair and protection proteins relates to overcoming stalled DNA replication that would otherwise cause growth arrest, rather than a response to oxidative damage. The importance of DNA repair for growth at low (subzero) temperatures was identified from proteomic analyses of the sea-ice bacterium Colwellia psychrerythraea, including a shift in cellular allocation of resources from DNA synthesis (initiated by DNA binding) to DNA repair (Nunn et al., 2015). CRISPR systems provide immunity against invading nucleic acids such as viruses (Sorek et al., 2013; Brendel et al., 2014), and Hht. litchfieldiae encodes a single type IB CRISPR system (Tschitschko et al., 2015). CRISPRassociated (Cas) proteins pertaining to all three stages of defence (adaptation, biogenesis, interference) had higher abundance at low temperature, including the Cas1 nuclease (halTADL1356) required for new spacer acquisition,


Cold adaptation of Antarctic haloarchaea 2219 and constituents of the Cascade complex: Cas5 (haltADL_1359); Cas6 (halTADL_1362), which is involved in the generation of CRISPR RNA; Cas7 (halTADL_1360), which forms the backbone of the Cascade complex; and Cas8b (halTADL_1361) (Sorek et al., 2013; Brendel et al., 2014). The proteome therefore indicates low temperature growth prompted the active surveillance and targeting by the CRISPR system. Extensive virus-host interactions and mechanisms of evasion, defence and adaptation have been described for the Deep Lake community (Tschitschko et al., 2015), so it is possible that the elevated functionality of the CRISPR system at low temperature enhances the ability of Hht. litchfieldiae to defend against invading viruses and remain the dominant species in its Antarctic habitat. Transcription, RNA turnover, translation and protein folding At low temperature, Hht. litchfieldiae had increased levels of RNA polymerase subunits while Hrr. lacusprofundi had decreased levels, indicating a fundamentally different transcriptional response for the two species. For Hht. litchfieldiae, several proteins associated with RNA degradation (Levy et al., 2011; Hou et al., 2014) also had higher abundance at low temperature: ribonuclease J (halTADL_2415) and the RNA-binding DnaG protein (halTADL_0764). This extended to an archaeal homolog of eukaryotic initiation factor 5A (halTADL_2262) for which the precise function in RNA turnover is unclear, although the C-terminal region is structurally similar to the RNA chaperone CspA (Wagner and Klug, 2007). For Hrr. lacusprofundi, ribonuclease J (Hlac_2047) also had higher abundance at low temperature. However, both species did have predicted transcriptional regulators and nucleic acidbinding OB-fold proteins with elevated abundance at low temperature, including those involved in sugar metabolism (TrmB) (halTADL_1392; Hlac_1076), phosphate uptake (PhoU) (Hlac_2346), and oxidative stress (RosR) (halTADL_0352). The TrmB regulators may facilitate Nglycosylation at low temperature (see Growth, morphology and cell envelope proteins above), by regulating the expression of sugar metabolism genes and ensuring that monosaccharides are available for N-glycosylation of the S-layer (Kandiba and Eichler, 2014; Todor et al., 2014). These proteins involved in RNA turnover and regulation of gene expression conceivably play specific roles in controlling RNA abundance at low temperature. Several tRNA modifying enzymes had higher abundance at low temperature, including NADPH-dependent 7cyano-7-deazaguanine reductase (halTADL_1477) and tRNA-guanine transglycosylase (Hlac_2160), which are involved in the synthesis of archaeosine and tRNA pseudouridine synthase (Pus10) (halTADL_0784; Hlac_1937).

Archaeosine is an archaeal tRNA modification found at the joint of the D-arm and T-arm of tRNA (Watanabe et al., 1997; Phillips et al., 2008), and Hfx. volcanii mutants that lack the ability to synthesize archaeosine are viable but cold sensitive (El Yacoubi et al., 2009; Blaby et al., 2010). This suggests that the archaeosine modification is not essential for tRNA function, but necessary for maintaining flexibility and correct folding of tRNA at low temperature (Blaby et al., 2010). Pseudouridylation of tRNA and rRNA is thought to be required for RNA stability, as well as to enhance translational accuracy, and in Hfx. volcanii Pus10 was found to be primarily responsible for pseudouridine insertion at two specific tRNA positions (Blaby et al., 2011). From genomic analyses of M. burtonii, dihydrouridine was predicted to be important for facilitating tRNA flexibility at low temperature, and subsequent analyses identified high levels in M. burtonii tRNA although total abundance was not found to increase at low temperature (Noon et al., 2003). Pseudouridines were also detected in M. burtonii tRNA (Noon et al., 2003). While Hht. litchfieldiae and Hrr. lacusprofundi each possess one tRNAdihydrouridine synthase (halTADL_0705; Hlac_2335), the proteins were not detected in the proteome. Overall, the current proteomic data provide support for archaeosine and pseudouridine as potentially playing a growth temperature-dependent role in maintaining tRNA flexibility in Antarctic haloarchaea. Similar to RNA polymerase subunits, at low temperature ribosomal proteins and elongation factor 2 (EF2) (Supporting Information Tables S1 and S2) had higher levels in Hht. litchfieldiae and lower levels in Hrr. lacusprofundi. Dph2, which catalyzes the first step of diphthamide synthesis, also followed these trends, consistent with archaeal EF2 containing diphthamide (a post-translationally modified histidine residue) (Zhang et al., 2010). The response of Hht. litchfieldiae has similarities to M. burtonii which also had elevated levels of ribosomal proteins at low temperature (Williams et al., 2010l; 2011). An exception to the differing trends observed for the two haloarchaea was the abundance of the translation initiation factors aIF1A (halTADL_0004; Hlac_1849) and aIF2 (halTADL_0923; Hlac_1009), which were higher in both organisms at low temperature. These components collectively stimulate translation initiation (Londei, 2005) and have previously been found to have higher abundance during low temperature growth in M. burtonii and Psychrobacter arcticus (Bergholz et al., 2009; Williams et al., 2010; Lauro et al., 2011). Initiation is likely the most cold sensitive translation process, and hence rate limiting step, in protein synthesis (Williams et al., 2010; Lauro et al., 2011). Increased abundance of initiation proteins may enable the translation apparatus to be primed and ready to process mRNA before secondary structures form that stall protein synthesis. This may have additional significance in view of each


2220 T. J. Williams et al. species encoding Csp and single TRAM domain nucleic acid chaperones (Taha et al., 2016) and RNA helicases, but none having significant differential abundance. These data indicate that unlike M. burtonii where RNA helicase and single TRAM domain proteins have increased abundance at low temperature (Williams et al., 2011), these Antarctic haloarchaea do not rely on similar strategies across the growth temperature ranges that were tested. For chaperoning and re/folding of nascent and existing proteins, Hht. litchfieldiae and Hrr. lacusprofundi both possess one set of DnaK-DnaJ-GrpE chaperones, one prefoldin (a and b subunits) and three group II (Cpn60) chaperonins. In addition, Hht. litchfieldiae has two cyclophilin- and three FKBP-type peptidyl-prolyl cis-trans isomerases (PPIases), whereas Hrr. lacusprofundi has one cyclophilin- and two FKBP-type PPIases. Not all of the proteins were detected in the proteome (Supporting Information Tables S1 and S2), but the main abundance changes were higher levels at low temperature for prefoldin beta subunit halTADL_1114 and Hlac_0567, Cpn60 halTADL_0092 and cyclophilin-type PPIase Hlac_1668, and higher levels at high temperature for DnaK halTADL_0595 and Hlac_0682, and GrpE Hlac_1291. The high temperature increases for DnaK and/or GrpE could be in response to heat stress (Ting et al., 2010; Williams et al., 2011). However, on the whole this pattern of abundance suggests that 4, 10 and 308C did not prompt major shifts in folding or refolding of proteins, indicating the cells were not particularly stressed (Ting et al., 2010; Williams et al., 2011). Hrr. lacusprofundi did have higher abundance at low temperature of three (Hlac_0344, 0345, 2381) of five Hsp 20-type chaperones, whereas abundance of the four from Hht. litchfieldiae was unchanged. In Sulfolobus solfataricus, low and high temperature growth and cold shock has been reported to increase mRNA levels of S.so-HSP20, with the purified protein found to inhibit aggregation of thermally denatured enzymes (Li et al., 2012). It is possible that Hrr. lacusprofundi deploys Hsp20 proteins to prevent improper folding of nascent polypeptides in order to maintain protein functionality at low temperature. These increases for Hsp20 mirror those for UspA (see Maintaining genome integrity and CRISPR defence above) and point to specific mechanisms by Hrr. lacusprofundi to facilitate growth at low temperature. Conclusion Hht. litchfieldiae and Hrr. lacusprofundi share a number of strategies for growing under conditions that approximate their natural environment (low temperature and high salt) (Fig. 5). While it is known that cell membranes can have a higher proportion of unsaturated lipids at low temperature to help maintain membrane fluidity (Nichols et al., 2004; Gibson et al., 2005; Cavicchioli, 2006), in order to maintain

osmotic balance (Dannenmuller et al., 2000; Oren, 2002; Oren, 2008; Dawson et al., 2012) our data suggest that for psychrophilic haloarchaea this requires higher abundances of MscS channels and Na1/H1 antiporters. The proteomic data would be consistent with both species undergoing extensive cell envelope changes, which in view of similar observations being made for M. burtonii (Campanaro et al., 2011; Williams et al., 2011), suggests this may be a characteristic of psychrophilic archaea. In addition to compositional changes, alterations to glycosylation of the Slayer also appear linked to low temperature adaptation. However, this is an area that will require further investigation as limited knowledge exists as to how alteration of Nglycosylation enables cells to adapt to different environmental conditions (Guan et al., 2012; Kaminski et al., 2013). Both species also exhibited evidence of enhanced capacities for RNA turnover, and tRNA modification that may promote tRNA low temperature flexibility. Molecular changes aimed at maintaining the correct structures of mRNA and initiating translation were responses shared by both Antarctic haloarchaea, and similar to lipid unsaturation, appear to be traits generally shared by psychrophiles (Siddiqui et al., 2013). In contrast, in view of the association of Csps with cold adaptation of bacteria and single TRAM domain proteins with archaea (Taha et al., 2016), it is interesting that neither class of proteins were associated with temperature-dependent growth, which may indicate that whatever functions they serve is not rate-limiting for growth down to 48C. A number of facets of the responses of Hht. litchfieldiae and Hrr. lacusprofundi set them apart, and some aspects may contribute to the dominance of Hht. litchfieldiae in Deep Lake. Stalled replication leading to DSBs may be an €m important barrier to growth at low temperature (Nystro and Neidhardt, 1994) and both species showed responses to counteract this impediment by increasing the cell’s capacity to protect DNA and repair DSBs. However, the strategies of the two species emphasize different processes, including the roles of UspA and Rad50. Hsp20 chaperones were also an unusual feature specific to Hrr. lacusprofundi. In addition, Hrr. lacusprofundi exhibited a shift to rod-shaped cells and formation of biofilms, with the latter possibly contributing to the species capacity to support high levels of gene exchange in Deep Lake (Madsen et al., 2012; DeMaere et al., 2013). The elevated CRISPR response of Hht. litchfieldiae may also promote its fitness in Deep Lake, as viruses are known to be prevalent in Deep Lake and capable of infecting several genera (Tschitschko et al., 2015). The higher abundances of glycerol kinase for Hht. litchfieldiae and Hrr. lacusprofundi at low temperature suggest that both species were metabolically poised to utilize glycerol, which is proposed to be a crucial growth substrate in



Fig. 5. Physiological responses of Hht. litchfieldiae and Hrr. lacusprofundi to growth at 48C, 108C or 308C. Depiction of the main cellular processes influenced by growth at high (308C, red) and low (108C and 48C, blue) temperature for Hht. litchfieldiae (A) and Hrr. lacusprofundi (B). Cellular processes or proteins common to both Hht. litchfieldiae and Hrr. lacusprofundi (green asterisk); CRISPR, clustered regularly interspaced short palindromic repeats system (type I-B); Dbp, DNA-binding protein; ABC, ATP-binding cassette transporter; TRAP, tripartite ATP-independent periplasmic transporter; MscS, mechanosensitive ion channel; NhaP, Na1/H1 antiporter; BCAAs, branched-chain amino acids; LTP, lamin tail protein; GlpK, glycerol kinase; G3P, glycerol 3-phosphate; G3PDH, glycerol 3phosphate dehydrogenase; DhaK, dihydroxyacetone kinase; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; PfdB, prefoldin subunit beta; GrpE, protein chaperone; DnaK, protein chaperone; PhoU, phosphate uptake regulator; TrmB, transcriptional regulator for sugar; DtxR, iron (metal) dependent transcriptional regulator (repressor); RNase J, ribonuclease J; DnaG, archaeal RNA-binding and degradation protein; Hsp20, heat shock protein 20; UspA, universal stress protein A; CetZ1 and CetZ2, archaeal cell shape control protein; PAPS, phosphoadenosine phosphosulfate; PAPSR, PAPS reductase; SqdB, UDP-sulfoquinovose synthase; RNAP, DNA-directed RNA polymerase; Ppa, inorganic pyrophosphatase; Cyc, cytochrome P450; CoxB, cytochrome c oxidase, subunit II; FtsZ, cell division protein; FtsA, cell division protein; M14, peptidase M14 carboxypeptidase; Sod, manganese/iron superoxide dismutase; TrxR, thioredoxin reductase; TAT, protein with twin-arginine translocation signal peptide; Signal P, protein with signal peptide.


2222 T. J. Williams et al. Deep Lake (Williams et al., 2014; Tschitschko et al., 2016). However, higher abundance of glycerol-3-phosphate dehydrogenase for Hht. litchfieldiae suggests an enhanced ability to direct glycerol to other processes via the key metabolic intermediate dihydroxyacetone phosphate. The fate of pyruvate, which is posited to be an important carbon source in hypersaline habitats (Oren, 2015), differed between the two species at low temperature: Hht. litchfieldiae appeared to utilize this substrate principally for generating biomass and reducing power via the TCA cycle, whereas Hrr. lacusprofundi seemed to divert a higher proportion of pyruvate toward carbon storage. It is therefore possible that the relative success of Hht. litchfieldiae in Deep Lake could partly be due to an enhanced ability to maintain cell replication and convert metabolic substrates under the prevailing Antarctic conditions. An important observation was the overall elevated levels of the core gene expression machinery in Hht. litchfieldiae compared to Hrr. lacusprofundi, which is consistent with Hht. litchfieldiae growing more rapidly under laboratory conditions at 48C (Fig. 1). The increased capacity may compensate for reduced rates of enzyme activity and increased stability of nucleic acid structures at low temperature. Hht. litchfieldiae is the most abundant species in Deep Lake and a number of traits, including trophic distinctions, have been identified as potential reasons for its dominance (DeMaere et al., 2013; Williams et al., 2014; Tschitschko et al., 2016). Beyond the genomic potential (DeMaere et al., 2013; Williams et al., 2014), these proteomic data provide evidence that temperature-dependent regulation of core transcriptional and translational processes are likely to facilitate cold adaptation of Hht. litchfieldiae and enhance its ecological competitiveness in Deep Lake. The new data lead us to hypothesize that Hrr. lacusprofundi enters a maintenance or dormant state in Deep Lake at a higher temperature than Hht. litchfieldiae. Future assessment of in situ growth rates of each species would enable this hypothesis to be tested.

Experimental procedures Growth of Antarctic haloarchaea Hht. litchfieldiae tADL and Hrr. lacusprofundi ACAM34 were grown in batch cultures in DBCM2 basal salt medium, 180 g L21 NaCl, 0.25 g L21 peptone and 0.05 g L21 yeast extract (Burns and Dyall-Smith, 2006), and supplemented with 10 mM sodium pyruvate as carbon source and 5 mM ammonium chloride as nitrogen source. Cultures were inoculated 1:100 from cultures grown under the same conditions in 50 mL medium in 250 mL flasks at 4, 10 and 308C at 120 rpm. Spectrophotometric readings at 600 nm were taken periodically to determine growth curves, using DBCM2 basal salt media as a blank.

Quantitative proteomics Proteomics was performed based on methods previously described for 8-plex iTRAQ proteomics of Hrr. lacusprofundi (Liao et al., 2016b). After cells reached mid-logarithmic phase, half of the culture volume was removed and cells harvested to obtain a whole cell pellet and an extracellular fraction (supernatant). A total of four biological replicates were prepared for each growth condition, with two of the replicates from each growth condition used for each 8-plex iTRAQ labelling run. The approach was used for proteins from whole cell extracts and the extracellular fraction, and two labelling experiments were performed for each fraction. This resulted in a total of eight 8-plex iTRAQ labelling experiments and a total of 64 protein samples analyzed for each strain, representing 4 vs. 30 8C and 10 vs. 30 8C. A TripleTOF 5600 1 hybrid tandem mass spectrometer (AB Sciex, Foster City, USA) operating in information-dependent acquisition mode using Analyst TF 1.7 software (AB Sciex, Foster City, USA) was used for sample analyses. Each 8-plex iTRAQ labelling experiment was run twice to provide two technical replicates. By running technical replicates of each 8-plex iTRAQ labelling experiment, a total of four datasets were generated for whole cell extracts and extracellular fractions of each growth condition. Each of the four datasets for the specific growth condition and fraction were combined and searched with ProteinPilot software 4.5 (AB Sciex, Foster City, USA) against the local Hht. litchfieldiae and Hrr. lacusprofundi protein FASTA database to identify proteins. A minimum unused score of 1.3 was accepted for protein identification, representing 95% confidence in correct sequence identification. Note that the number of detected proteins using an unused score 1.3 was less than that detected using a 1% or 5% false discovery rate (Supporting Information Table S3). For quantitative analysis of relative abundance level changes, data were considered statistically significant when p was less than 0.05 and the error factor was less than 2. The weighted average mean and standard deviation of differential abundance between iTRAQ reporter ion ratios were calculated (Zhou et al., 2007). In addition, an average weighted abundance ratio of 1.5-fold or more was used as the cutoff for differential abundance of proteins from 4 vs. 308C, and 10 vs. 308C cultures. Pearson’s correlation analysis between biological replicates, technical replicates and labelling experiment replicates was performed using SPSS 22.0 software (Supporting Information Fig. S3). In some cases, proteins with 1.2 – 1.5 fold differential abundance were considered if they were from functional categories represented by the core set of proteins (Supporting Information Tables S1 and S2). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaıno et al., 2014) with the dataset identifier PXD005076. All proteins of relevance were manually functionally annotated as previously described (Liao et al., 2016b). Complete details are provided in Supplementary information.

Microscopy analysis of cell morphology and PHA Hht. litchfieldiae and Hrr. lacusprofundi cells grown to midlogarithmic phase at different temperatures (30, 10 and 48C) and fixed with 2% glutaraldehyde were examined using a


Cold adaptation of Antarctic haloarchaea 2223 JEOL 7001F field emission scanning electron microscope (JEOL, Freising, Germany) as previously described (Liao et al., 2016b). Scanning electron microscopy was performed under high-vacuum mode, the working distance of detectors from the specimen was set to 10 mm, and the accelerating voltage was applied at 15 kV. PHA was assessed by staining cells with Nile Blue A (Ostle and Holt, 1982), a fluorescent dye that can be diagnostic for PHA granules in both bacteria and archaea (e.g. Legat et al., 2010; Bhuwal et al., 2013; Mesquita et al., 2015). Cells from logarithmic phase at different growth temperatures were stained with Nile Blue A based on the methods described for haloarchaeal species (Legat et al., 2010) with minor modifications. A 1% aqueous solution of Nile Blue A (Sigma-Aldrich, St. Louis, USA) was filtered and mildly heated (378C) before use. The cells (10 lL) were dried on a glass slide. The heated-fixed smears of cells were then stained with 5 lL Nile Blue A solution at 558C on a heating block for 10 min, followed by washing with tap water for 1 min, 8% acetic acid for 1 min, and tap water again for 1 min; finally the stained smear was dried at room temperature and covered with a glass cover slip. Samples were examined with a digital microscope system (Olympus BX61 microscopy with DP71 camera; Olympus, Tokyo, Japan) using both using bright-field and fluorescencefield imaging (Olympus WIBA filter), as described previously (Liao et al., 2016a). Transmission electron microscopy was used to visualize PHA-like granules in Hrr. lacusprofundi cells grown at 48C. Logarithmic phase cells (10 mL) were pelleted by centrifugation for 20 min at 4,500 x g, and the cell pellet washed with 3 x DBCM2 salt solution. Cell pellets were fixed for 1 h at room temperature in 4% w/v paraformaldehyde, 2.5% glutaraldehyde and 0.3 M sucrose in 0.1 M cacodylate buffer (Sigma–Aldrich, pH 7.2). Fixed cells were centrifuged at 5,000 x g for 10 min at room temperature and the supernatant was aspirated. Pellets were re-suspended in 0.1 M cacodylate buffer, washed for 5 min, and the cells recovered by centrifugation at 5,000 x g for 10 min, with the process performed twice. The cells were embedded in 2% w/v agar, washed with 0.1 M cacodylate buffer and postfixed in 1% w/v osmium tetroxide (OsO4, in 0.1 M cacodylate buffer pH 7.2) for 1 h. After post-fixation, the samples were washed with MilliQ water and dehydrated through a graded ethanol series (50–100%), and the samples were embedded in L.R. White resin (80% polyhydroxy substituted bisphenol, a dimethacrylate resin 19.6%, C12 methacrylate ester, 0.9% benzoyl peroxide) and oven-dried in a gelatin capsule at 608C for 24 h. Ultrathin sections were cut using a Lecia EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and mounted onto cleaned 200 copper-mesh formvar-coated grids. The sections were examined with a JEOL 1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan).

Conflict of Interest The authors declare no conflict of interest. Acknowledgements This work was supported by the Australian Research Council [DP150100244]. YL and JY were funded by the China Scholarship Council (File No. 201206910027 and 201206230085,

respectively). Mass spectrometry results were obtained at the Bioanalytical Mass Spectrometry Facility and electron microscopy at the Electron Microscope Unit, both within the Analytical Centre of the University of New South Wales. Subsidized access to these facilities is gratefully acknowledged. We thank the PRIDE team and ProteomeXchange for efficiently processing and hosting the mass spectrometry data. We acknowledge the insightful and constructive comments provided by the reviewers during the review process.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig. S1. Fluorescence microscopy analysis of PHA in Hrr. lacusprofundi. Fig. S2. Predicated protein domains of Hlac_1732 compared to Haloferax mediterranei PHA synthase PhaC subunit. Fig. S3. Statistical analyses of iTRAQ data from repeat experiments. Table S1. Halohasta litchfieldiae and Halorubrum lacusprofundi proteins that showed temperature-dependent differential abundance. Table S2. Complete list of temperature-dependent, differentially abundant proteins from Halohasta litchfieldiae and Halorubrum lacusprofundi. Table S3. The number of detected proteins using unused score 1.3, FDR 1%, and FDR 5%. nd, not determined.
