Glycerol metabolism of haloarchaea - Wiley Online Library

Environmental Microbiology (2017) 19(3), 864–877

doi:10.1111/1462-2920.13580

Glycerol metabolism of haloarchaea

Timothy J. Williams, Michelle Allen, Bernhard Tschitschko and Ricardo Cavicchioli* School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, New South Wales, Australia. Summary Haloarchaea are heterotrophic members of the Archaea that thrive in hypersaline environments, often feeding off the glycerol that is produced as an osmolyte by eucaryotic Dunaliella during primary production. In this study we analyzed glycerol metabolism genes in closed genomes of haloarchaea and examined published data describing the growth properties of haloarchaea and experimental data for the enzymes involved. By integrating the genomic data with knowledge from the literature, we derived an understanding of the ecophysiology and evolutionary properties of glycerol catabolic pathways in haloarchaea.

Introduction Halophilic archaea (or haloarchaea; Halobacteria) live in environments with salt concentrations approaching saturation, including natural brines, salt lakes, the Dead Sea, marine solar salterns, salted fish and other high-salt foodstuffs, and salted hides (Soppa, 2006; Stan-Lotter and Fendrihan, 2015). The unicellular eucaryotic microalga Dunaliella (Chlorophyceae) is found worldwide in saltern evaporation and crystallizer ponds as well as in many natural salt lakes (Oren, 2009), although it is no longer found in the Dead Sea due to the increased salinity of this lake and the unfavourable ionic composition of the brine (Oren, 2014). Dunaliella is the main or sole primary producer in the hypersaline environments in which it grows, and produces molar amounts of intracellular glycerol (up to 7–8 M) as an osmolyte (Borowitzka, 1981; Oren, 1999; Elevi Bardavid et al., 2008). Glycerol is used for osmotic adaptation in halophilic eucaryotic algae and fungi, but not in bacteria and archaea due to the permeability of their respective

*For correspondence. E-mail [email protected]; Tel. 161 2 9385 3516; Fax: 161 2 9385 2742. C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd V

membranes to glycerol (Ben-Amotz and Avron, 1973; Ben-Amotz and Grunwald, 1981; Roberts, 2005; Oren, 2008). As a compatible solute, glycerol fulfils an osmotic role in Dunaliella by maintaining cell volume and protecting enzymes against inactivation under high-salt conditions. The cytoplasmic membrane of Dunaliella is mostly impermeable to glycerol, and excess production of glycerol can be converted into starch and mobilized later as a source of glycerol (Ben-Amotz and Avron, 1981; Elevi Bardavid et al., 2008). However, glycerol can be released as a result of leakage from healthy cells, hypotonic stress, and cell lysis (Oren and Gurevich, 1994a; Oren, 1999; Elevi Bardavid et al., 2008). The hyperthermophilic archaeon Archaeoglobus fulgidus utilizes the glycerol derivative diglycerol phosphate (DGP; 1,10 -diglyceryl phosphate) as an osmolyte under high-salt conditions (Martins et al., 1997; Lamosa et al., 2000). In contrast, haloarchaea principally adopt a “salt-in” strategy whereby molar concentrations of potassium and chloride ions are accumulated in the cytosol to maintain osmotic balance against high external sodium chloride concentrations (Oren, 2008). Under laboratory growth conditions, certain haloarchaea have been reported to either synthesize or import compatible solutes (e.g., 2-sulfotrehalose, glutamate, proline, glycine betaine) to supplement (Desmarais et al., 1997; Kokoeva et al., 2002) or possibly replace (Goh et al., 2011) the ‘salt-in’ strategy for osmoadaptation. However, while potassium transport proteins were initially reported to be absent from the stromatolite isolate Halococcus hamelinensis (Goh et al., 2011), subsequent genome sequencing revealed sequences for transporters that may be capable of importing potassium (Gudhka et al., 2015). Importantly, there is no evidence that haloarchaea employ glycerol (or any glycerol derivative) as a compatible solute; thus, glycerol produced by Dunaliella is used only as a carbon and energy source by haloarchaea. To date, over 50 genera of haloarchaea have been characterized, and the ability of axenic cultures to grow on glycerol has been demonstrated for the majority of these. Many haloarchaea are equipped with two pathways for glycerol catabolism (Fig. 1). Glycerol can either be phosphorylated by glycerol kinase to sn-glycerol-3-phosphate (G3P) which is then oxidized by G3P dehydrogenase (G3PDH) to dihydroxyacetone phosphate (DHAP), or glycerol can be oxidized by glycerol dehydrogenase to

Glycerol metabolism of haloarchaea 865

Fig. 1. Haloarchaeal pathways for the uptake and catabolism of glycerol, dihydroxyacetone, and glycerol-3-phosphate. ABC, ATP-binding cassette; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate.

dihydroxyacetone (DHA) which is then phosphorylated by DHA kinase to DHAP. The two alternative pathways are referred to here as the G3P pathway and DHA pathway, respectively. Both pathways can be found in glyceroldegrading bacteria, and may have been acquired by haloarchaea from bacteria as part of their evolutionary transformation to a heterotrophic lifestyle (Nelson-Sathi et al., 2012). One hypothesis is that glycerol kinase began as a primeval salvaging enzyme for intracellular glycerol associated with turnover of bacterial phospholipids, and was later co-opted as a scavenging enzyme for extracellular glycerol (Lin, 1976). Both the G3P and DHA pathways involve a physiologically irreversible phosphorylation step (glycerol kinase or DHA kinase), and a reversible oxidation step (glycerol dehydrogenase or G3P dehydrogenase). DHAP, the product of either pathway, is an important metabolic intermediate that can be directed to glycolysis (Embden-Meyerhof pathway) or gluconeogenesis, as well as being the immediate

precursor to sn-glycerol-1-phosphate (G1P), the backbone of archaeal phospholipids. Certain haloarchaea (including Haloferax spp. and Haloarcula spp.) grown in media containing glycerol release the organic acids pyruvate, acetate, and lactate as overflow products (Oren and Gurevich, 1994b); in haloarchaea, lactate can be generated from DHAP via the methylglyoxal bypass (Oren, 1994; Oren and Gurevich, 1995), as well as from pyruvate by lactate dehydrogenase (Hecht et al., 1990). Glycerol and its metabolic products pyruvate and DHA are preferred substrates for both archaea and bacteria in many hypersaline environments (Oren, 2015). Many haloarchaea likely have a metabolism that is oriented towards glycerol. Hfx. volcanii exhibits catabolite repression of glucose metabolism by glycerol, consistent with a metabolic preference for glycerol over sugars (Holtman et al., 2001). This contrasts with Escherichia coli, which exhibits diauxic growth with glucose as the preferred carbon and energy source over glycerol (Sherwood et al., 2009).

C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 19, 864–877 V

Fig. 2. Glycerol-related proteins encoded in haloarchaeal genomes. Each tick signifies a gene for the protein detected in one of the 32 specified genomes. Red and blue ticks signify that proteins for different enzymes or transporters are encoded in the same gene cluster; otherwise ticks are black. Proteins pertaining to the same enzyme complex or transporter system are denoted with the same colour. Location data for species and strains after Anderson et al. (2009), Antunes et al. (2008; 2011), Burns et al. (2004; 2007; 2010), Castillo et al. (2006; 2007), DeMaere et al. (2013), Ding et al. (2014), Dominova et al. (2013), Dyall-Smith et al. (2011; 2013), Bolhuis et al. (2006), Falb et al. (2005), Feng et al. (2012), Gruber et al. (2004), Gutierrez et al. (2007), Ihara et al. (1997), Juez et al. (1986), Liu et al. (2011), Lv et al. (2015), Malfatti et al. (2009), Messina et al. (2016), Montalvo-Rodrıguez et al. (1988), Mullakhanbhai and Larsen € per (1982), Sorokin et al. (1975), Ng et al. (2000), Oren et al. (1988; 2002), Pfeiffer et al. (2008), Roh et al. (2007; 2010), Saunders et al. (2010), Siddaramappa et al. (2012), Soliman and Tru (2016a,b), Tindall et al. (1980; 1984), Wainø et al. (2000), Yun et al. (2015), Zvyagintseva and Tarasov (1987). ABC, ATP-binding cassette; DHA, dihydroxyacetone; G3P, glycerol-3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; Haa., Halanaeroarchaeum; MCP-UgpB, protein of unknown function that comprises a methyl-accepting chemotaxis protein signalling domain (MCP) and G3P-binding domain (UgpB); MIP, Major Intrinsic Protein family; SBP, solute-binding protein.

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Glycerol metabolism of haloarchaea 867 In this study we surveyed 32 closed haloarchaeal genomes for the presence of genes implicated in glycerol catabolism in order to determine the genomic potential of different species to metabolize glycerol (Fig. 2). Genes examined included those involved in the conversion of glycerol to DHAP, and the uptake of glycerol or G3P. Phylogenetic analyses were also performed to assess the evolutionary relationship of specific genes, including the role of horizontal gene transfer in conferring specific traits (e.g. glycerol kinase). The study integrated knowledge of the genomic analyses with physiological and ecological findings reported in the literature, to generate a view of the role that glycerol plays in supporting the growth of haloarchaea in hypersaline environments. Glycerol uptake Cell membranes are intrinsically permeable to glycerol as a consequence of the small size of this molecule and its neutral charge, so glycerol can readily enter (and depart) the cell by simple diffusion (Richey and Lin, 1972). However, at low concentrations of glycerol, cells that are limited to simple diffusion have a growth disadvantage (Richey and Lin, 1972). In archaea and bacteria, several mechanisms exist to conduct glycerol into the cell. One mechanism is facilitated diffusion using a glycerol transporter (GlpF), a transmembrane channel protein (Major Intrinsic Protein [MIP] family) (Stroud et al., 2003; Anderson et al., 2011). The motive force for net glycerol uptake by diffusion results from the imbalance of intra- and extracellular glycerol concentrations caused by glycerol catabolism (Yeh et al., 2009). Thus, effective glycerol phosphorylation is dependent on the activity of both the facilitator and glycerol kinase (Voegele et al., 1993). Of the 32 haloarchaeal genomes surveyed (Fig. 2), only Halomicrobium mukohataei had an identifiable GlpF transporter (Anderson et al., 2011), with the gene for this transporter in the same gene cluster as the genes encoding glycerol kinase and G3PDH (Fig. 2). Instead of GlpF, most haloarchaeal genomes encode an uncharacterized transmembrane protein (Fig. 2) that was predicted to encode a new family of glycerol transporters, based on the gene being adjacent to a gene for glycerol kinase in haloarchaeal genomes (Anderson et al., 2011). Among the haloarchaeal genomes surveyed, this putative glycerol transporter is only found in genomes that also encode glycerol kinase, and at least one copy is invariably found adjacent to a glycerol kinase gene. Some haloarchaeal genomes possess multiple copies of this putative transporter (Fig. 2). Members of this new family are also widespread among bacterial genomes, where they are often found adjacent to genes involved in glycerol or propanediol metabolism (e.g., glycerol/propanediol dehydratase, glycerol kinase) (Anderson et al., 2011; see Glycerol dehydratase (EC 4.2.1.30) below). In the Antarctic

haloarchaeon strain DL31 (unknown genus closely related to Halolamina), a potential G3P pathway is encoded but it lacks any identifiable glycerol transporter. It was hypothesized that strain DL31 obtained glycerol in its lake habitat via simple diffusion by direct association with Dunaliella cell material (Williams et al., 2014). However, of the haloarchaea surveyed here, DL31 is the only one to encode a glycerol kinase but not a GlpABC-type G3PDH (Fig. 2). A role for the GlpA2-type G3PDH in glycerol catabolism has yet to be established (see G3PDH (EC 1.1.5.3) below), which raises the possibility that DL31 does not possess a functional glycerol metabolism. Certain bacteria (e.g., Rhizobiaceae) utilize a glycerol ABC transporter system for the uptake of glycerol, which is likely to scavenge very low concentrations of glycerol present in soil and rhizosphere environments (Ding et al., 2012). No such primary transport systems for glycerol are known for haloarchaea, and may not be necessary in hypersaline environments in which glycerol is supplied by Dunaliella. Glycerol kinase (EC 2.7.1.30) Once transported into the cell, glycerol can be phosphorylated by glycerol kinase and trapped inside the cell as G3P. Glycerol kinase is an ATP-dependent homotetrameric enzyme (de Riel and Paulus, 1978) with high-affinity for glycerol, and is proposed to allow organisms to grow effectively in environments containing relatively low concentrations of glycerol (Neijssel et al., 1975). For a range of tested haloarchaeal species, glycerol kinase activity was shown to be constitutive (Oren and Gurevich, 1994a). Glycerol kinase is capable of phosphorylating both glycerol and DHA (Lin, 1976; Ouellette et al., 2013). In Hfx. volcanii, glycerol kinase was shown to be more important than DHA kinase for growth on DHA; deletion of either the glycerol kinase gene (glpK) or the DHA kinase operon (dhaKLM) both led to reduced growth on DHA, but the reduction was more pronounced for the glpK mutant (Ouellette et al., 2013). It is unknown if this was due to higher expression levels of glycerol kinase compared to DHA kinase, or to a higher affinity of glycerol kinase compared to DHA kinase for DHA (Ouellette et al., 2013); the former may be more likely given that, in bacteria, DHA kinase has higher affinity for DHA compared to glycerol (Lin, 1976). The phylogeny of haloarchaeal glycerol kinase protein sequences differs to the phylogeny of the organisms based on their 16S rRNA genes (Fig. 3), indicating that multiple horizontal gene transfer events contributed to the distribution of glycerol kinase genes among haloarchaea. The glycerol kinase genes of haloarchaea are usually found in the same gene clusters as genes for a putative glycerol transporter (Anderson et al., 2011) and GlpABC-type G3PDH (Fig. 2), which suggests that this glycerol-related gene cluster was acquired as a unit. In some species,


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Fig. 3. Phylogenetic analysis of haloarchaeal glycerol kinase proteins and representative 16S rRNA genes. Thirty-one glycerol kinase genes were identified from the complete haloarchaeal genomes by BLAST (Altschul et al. 1990) using GlpK from Hrr. lacusprofundi (Hlac_1122) as the query. To facilitate comparison of glycerol kinase gene phylogeny with taxonomic (16S rRNA gene-based) relationships among the Halobacteria, representative 16 rRNA genes were selected. Multiple alignments of GlpK protein sequences and 16S rRNA nucleotide sequences were created using MUSCLE (Edgar, 2004) and used to construct phylogenetic trees by the Maximum-likelihood method in MEGA6 (Tamura et al., 2013). For the GlpK tree, all amino acid positions with less than 80% site coverage were eliminated, resulting in 510 amino acids in the final dataset. The 16S rRNA gene tree included all positions, with 1413 nucleotide positions in the final dataset. The trees were rooted by E. coli strain K12 MG1655 GlpK and E. coli strain K12 MG1655 rrnaA, respectively (not shown). In each case the tree with the highest log likelihood is shown. For each tree, 1000 bootstraps were performed and bootstrap values greater than 70% are reported. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Genes are labelled with their IMG locus tag and species name, with colouring highlighting the position of corresponding taxa or groups. An asterisk marks those completed genomes in which a GlpK protein was not detected.

this gene cluster also includes DHA kinase genes (Fig. 2). Three haloarcheal species surveyed have multiple glycerol kinase genes: Halohasta litchfieldiae (3 genes), Halogeometricum borinquense (2) and Natronomonas moolapensis (2) (Fig. 2). Their phylogeny suggests that the two in Nmn. moolapensis were most likely acquired independently, but for Hht. litchfieldiae and Hgm. borinquense the genes appear to have arisen as a result of duplication within the genome (Fig. 3). G3P uptake In addition to being an intermediate in the first pathway of glycerol dissimilation, G3P serves as an important

environmental substrate in its own right. G3P is a ubiquitous constituent of cellular material, including the backbone of (non-archaeal) phospholipids, which is readily liberated during biological decomposition, and is chemically stable (Lin, 1976). Further, Dunaliella exhibits enhanced turnover of specific phospholipids in response to osmotic changes (Chitlaru and Pick, 1991), so levels of glycerol and glycerophosphodiesters in hypersaline environments may be linked. Most of the surveyed haloarchaeal genomes encode G3P ABC transporter systems (UgpBAEC) for primary transport of G3P into the cell (Fig. 2). These are members of the bacterial CUT 1 subfamily of ABC transporters, which include transporters for the uptake of carbohydrates and polyols (Schneider, 2001;


Glycerol metabolism of haloarchaea 869 Wuttge et al., 2012). In bacteria, G3P transporters are also used for primary transport of G3P diesters into the cell (Wuttge et al., 2012). In haloarchaea, the gene cluster that encodes the UgpBAEC ABC transporter was often associated with a gene for glycerophosphodiester phosphodiesterase (EC 3.1.4.46), a hydrolytic enzyme that has broad specificity for glycerophosphodiesters and releases G3P. Thus, these haloarchaea are likely equipped to both import and degrade a broad range of glycerophosphodiesters. However, although G3P can be derived from bacterial and eucaryotic phospholipids, it is not a constituent of archaeal phospholipids (see G1P dehydrogenase (EC 1.1.1.261) below). In bacteria, G3P transporters (GlpT) of the Major Facilitator Superfamily (MFS) can also uptake G3P into the cell (Law et al., 2008). These secondary transporters couple the outward flow of internal inorganic phosphate to the uptake of G3P (Law et al., 2008). While MFS proteins were identified in the haloarchaeal genomes surveyed, their sequence identities do not support a function in G3P uptake. G3PDH (EC 1.1.5.3) In bacteria, heterotrimeric G3PDH, composed of the subunits GlpA, GlpB and GlpC, and a homodimeric G3PDH (GlpD) are both used for the oxidation of G3P to DHAP; both enzymes also function in respiration (Ingledew and Poole, 1984). GlpD (‘aerobic G3P dehydrogenase’) catalyzes the oxidation of G3P to DHAP, with reducing equivalents ultimately passed on to oxygen or nitrate, and both soluble and membrane-bound isoforms of GlpD are known (Yeh et al., 2008). GlpD is homologous to GlpA but lacks a C-terminal BFD (bacterioferritin-associated ferredoxin)-like [2Fe-2S]-binding domain present in GlpA. The oxidation of G3P to DHAP by heterotrimeric G3PDH (GlpABC; ‘anaerobic G3P dehydrogenase’), a membraneassociated enzyme, is coupled to the reduction of fumarate or nitrate, with concomitant translocation of protons to generate a proton motive force (Kistler and Lin, 1971; Ingledew and Poole, 1984). The presence of a BFD-like domain suggests this protein may serve as a general redox enzyme and/or in iron metabolism in addition to G3P oxidation (Garg et al., 1996; Rawls et al., 2011). In Hfx. volcanii, GlpABC functions during aerobic growth on glycerol, and the genes are cotranscribed (Sherwood et al., 2009; Rawls et al., 2011). The Hfx. volcanii genome encodes two copies of GlpABC, the only haloarchaeon surveyed for which this is the case (Fig. 2). Analysis of Hfx. volcanii knockout strains revealed that only one copy (the glpABC located next to glpK on the primary replicon) is required for growth on glycerol (Rawls et al., 2011). The levels of transcripts for glpABC and glpK were significantly upregulated in aerated cultures in the presence of glycerol

(Sherwood et al., 2009). Although the regulatory mechanism is unknown, individual glpA- and glpK-specific transcripts were 78- and 9-fold more abundant in glycerol versus glucose-grown cultures, respectively (Sherwood et al., 2009; Rawls et al., 2011). In E. coli, G3P is an inducer that binds GlpR (DeoR-type transcriptional regulator) to derepress the glycerol metabolic regulon (Zeng et al., 1996). However, in Hfx. volcanii GlpR is not required for regulation of GlpABC G3PDH gene expression, and regulation is assumed to be mediated by an as yet uncharacterized regulator (Rawls et al., 2011). Instead in Hfx. volcanii, GlpR regulates expression of both fructose and glucose metabolic genes through repression of pfkB (phosphofructokinase) and kdgK1 (2keto-3-deoxy-D-gluconate kinase) (Rawls et al., 2010). The phylogenetic analysis of haloarchaeal GlpA shows that sequences have a common origin, with the exception of one Halorhabdus utahensis GlpA (Huta_0683) that is likely a more recent acquisition from bacteria, probably a halophilic member of the Clostridia (Huta_0683: 68% match to Halanaerobium saccharolyticum GlpA) (Fig. 4). Also present across the haloarchaeal genomes is a GlpA homolog (GlpA2) that, like bacterial GlpD, can be distinguished from the GlpA subunit of the GlpABC complex by the absence of a C-terminal BFD-like domain (Rawls et al., 2011). Phylogenetic analysis of haloarchaeal GlpA and GlpA2 shows they form a cluster that excludes both bacterial GlpA and GlpD proteins (Fig. 4). Rather than haloarchaeal GlpA2 and bacterial GlpD sharing a common origin, the most parsimonious interpretation is that the haloarchaeal GlpA2 arose through the loss of the C-terminal BFD-like domain from haloarchaeal GlpA. GlpABC is encoded by most haloarchaea that also encode glycerol kinase, and GlpA2 also has a broad distribution (Fig. 2). However, Halobacterium sp. strain DL1 has GlpA2 and Halanaeroarchaeum sulfurireducens has GlpABC, but neither species possess other glycerolrelated proteins including those for G3P uptake (Fig. 2), suggesting that G3PDH plays no role in glycerol metabolism in these strains. Halobacterium sp. strain DL1 is an Antarctic strain that is characterized by a preference for catabolism of amino acids (especially branched chain amino acids), and does not utilize glycerol (Williams et al., 2014), and Haa. sulfurireducens is a close relative of Halobacterium but has a very limited carbon metabolism (Sorokin et al., 2016a,b). The GlpA2 protein from Halobacterium sp. strain DL1 clusters with GlpA2 from other Halobacterium spp. which contain the full G3P pathway for glycerol catabolism. The data suggest that Halobacterium sp. strain DL1 and Haa. sulfurireducens may be in the process of losing glycerol catabolism genes, or the genes they possess (GlpA2 or GlpABC) perform functions unrelated to glycerol metabolism. In general, more needs to be learned about the roles of apparent ‘relic’ components and


870 T. J. Williams et al. the functional properties of GlpA and GlpA2 in G3PDH activity in order to better understand their roles in glycerol metabolism in haloarchaea.

For completeness, it should be noted that G3PDH (both GlpABC and GlpA2) is distinct from the GpsA-type G3PDH of the thermophilic archaeon A. fulgidus, which


Glycerol metabolism of haloarchaea 871 Fig. 4. Phylogenetic analysis of G3P dehydrogenase alpha subunit GlpA and its homologs GlpD and GlpA2 across Archaea and Bacteria. GlpA (blue) and GlpA2 (orange) protein sequences were identified in the complete haloarchaeal genomes by BLAST (Altschul et al., 1990) using GlpA from Hrr. lacusprofundi (Hlac_1123) and GlpA2 from Halohasta litchfieldiae tADL (HalTADL_2244) as queries. A multiple alignment of these sequences, together with selected bacterial (GlpD, green) and eucaryotic homologs, was created using MUSCLE (Edgar, 2004) and used to construct phylogenetic trees by the maximum-likelihood method in MEGA6 (Tamura et al., 2013). The tree with the highest log likelihood is shown. All amino acid positions with less than 80% site coverage were eliminated, resulting in 393 amino acid positions in the final dataset; where present in the protein sequence, this represents the region upstream of the C-terminal BFD (bacterioferritin-associated ferredoxin)-like [2Fe-2S]-binding domain. The tree is drawn to scale with branch lengths measured in the number of substitutions per site, and rooted by the eucaryotic sequences Saccharomyces cerevisiae GlpA and mitochondrial Mus musculus Gpd2. For each tree, 1000 bootstraps were performed and bootstrap values greater than 70% are reported.

possibly serves in the synthesis of the compatible solute DGP (Lamosa et al., 2000; Sakasegawa et al., 2004); this enzyme is not encoded in any of the haloarchaeal genomes. G1P dehydrogenase (EC 1.1.1.261) G1P dehydrogenase (EgsA) catalyzes the reversible conversion between DHAP and G1P. A gene for G1P dehydrogenase was identified in all the haloarchaeal genomes surveyed. G1P dehydrogenase is an essential enzyme in archaea because it generates the glycerophosphate backbone (G1P) of archaeal diether phospholipids (Nishihara et al., 1999). Whereas bacteria and eucaryotes contain D-glycerol in their cell membranes, archaea possess L-glycerol in their cell membranes, and the G1P of archaeal phospholipids is linked to isoprenyl side chains using highly stable ether bonds (White et al., 1996). These bonds would be resistant to phosphodiesterase (see G3P uptake, above), and an archaeal mechanism for cleaving ether bonds to release G1P has not been described. Thus, it is unknown if turnover and biodegradation of archaeal phospholipids could liberate glycerol and/or phosphate for reuse by haloarchaeal cells. Glycerol dehydrogenase (EC 1.1.1.6) Haloarchaeal glycerol dehydrogenase belongs to the ‘ironcontaining’ (Fe21 or Zn21) alcohol dehydrogenase family (Ruzheinikov et al., 2001; Falb et al., 2008) and appears to perform a peripheral role in glycerol metabolism. Glycerol dehydrogenase genes are present in far fewer (nine) haloarchaeal genomes than glycerol kinase genes (27) (Fig. 2), and their presence/absence in genomes is consistent with glycerol dehydrogenase activity (NAD1dependent) being demonstrated for Hbt. salinarum but not for Haloferax and Haloarcula spp. (Oren and Gurevich, 1994a). The glycerol dehydrogenase genes were never located within or close to gene clusters encoding other glycerol catabolism genes or DHA kinase genes. Glycerol dehydrogenase has low affinity for glycerol (unlike glycerol kinase, which has high affinity), leading to the proposal (based on Klebsiella aerogenes) that the enzyme functions when glycerol concentrations are relatively high (Tempest

and Neijssel, 1981). In Hbt. salinarum R1 the glycerol dehydrogenase gene is regulated by the Lrp transcriptional regulator (Schwaiger et al., 2010). As a low-affinity enzyme, glycerol dehydrogenase in haloarchaea might be utilized when environmental levels of glycerol are high, such as during the decay phase of a Dunaliella bloom, to ensure utilization of abundant glycerol and reduce the possibility of substrate inhibition affecting cell growth. However, in certain environments glycerol concentrations may rarely be high enough to utilize a low-affinity glyceroldegrading enzyme. DHA kinase (EC 2.7.1.29) Glycerol kinase acts on glycerol and DHA, whereas DHA kinase catalyzes the conversion of DHA, D-glyceraldehyde, and possibly other short-chain aldehydes and ketones, but not glycerol (Zurbriggen et al., 2008). Haloarchaeal DHA kinase consists of three subunits: DhaK, DhaL, DhaM (Erni et al., 2006). DHA is posited to be a potentially important substrate in hypersaline environments, although not in highly alkaline environments where DHA is unstable (Lin, 1976). It may be generated as an overflow product of the glycerol cycle in Dunaliella (Elevi Bardavid and Oren, 2008; Ouellette et al., 2013), or by incomplete glycerol oxidation in the halophilic bacterium Salinibacter ruber (Sher et al., 2004; Elevi Bardavid and Oren, 2008), which is the dominant bacterium in crystallizer ponds (Mongodin et al., 2005). Nine of the haloarchaeal genomes encode DHA kinase, but only four of these also encode glycerol dehydrogenase, which suggests that exogenous DHA is used as a substrate and phosphorylated to DHAP. Hqr. walsbyi can use DHA as a carbon and energy source (Bolhuis et al., 2006; Elevi Bardavid and Oren, 2008), and growth has been observed to benefit from the presence of S. ruber in culture (Bolhuis et al., 2004); thus, it has been suggested that this is a synergistic effect caused by the release of DHA by S. ruber (Bolhuis et al., 2006; Elevi Bardavid and Oren, 2008). Hqr. walsbyi is one of the haloarchaeal taxa that have DHA kinase but lack an identifiable glycerol dehydrogenase, consistent with DHA being an important exogenous substrate, since (in the


872 T. J. Williams et al. absence of glycerol dehydrogenase) it cannot be generated endogenously by glycerol oxidation. A cytosolic phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS) is responsible for the phosphorylation of DHA to DHA phosphate (DHAP). Phosphoryl flow (initially at the expense of PEP) proceeds from the general PTS proteins EI and Hpr to the DHA kinase component DhaM, which is homologous to EIIA of transmembrane ATP-dependent PTS (Gutknecht et al., 2001). Translocation of DHA across the membrane occurs via facilitated diffusion, driven by a concentration gradient maintained by cytosolic phosphorylation of DHA (Bolhuis et al., 2006). No DHA transporter is known, although in certain bacteria, facilitated diffusion of extracellular DHA is thought to occur using the same facilitator as glycerol uptake (e.g., GlpF) (Monniot et al., 2012). In Haloferax spp., Hht. litchfieldiae and Salinarchaeum sp., the DHA kinase genes are located close to the glycerol catabolism gene cluster (Fig. 2), along with the gene for the phosphocarrier protein Hpr of the PEP-PTS system (Haloferax. spp) or the genes for the complete PEP-PTS system (Hht. litchfieldiae, Salinarchaeum sp.). Genomes that encode DHA kinase genes always also contain PEP-PTS genes, although the converse is not always true: PEP-PTS can be used for the phosphorylation and translocation of sugars, not just glycerol. One evolutionary possibility is that the ability to utilize DHA as a substrate arose as a consequence of the catabolic pathway initiated by glycerol dehydrogenase (Lin, 1976). However, whereas G3PDH is always encoded in haloarchaeal genomes that encode glycerol kinase, the same is not true for DHA kinase and glycerol dehydrogenase. Hbt. salinarum encodes a predicted glycerol dehydrogenase but no DHA kinase (Falb et al., 2008; Anderson et al., 2011). This is a feature of several haloarchaeal genomes (Fig. 2), and it is possible that DHA is phosphorylated to DHAP by glycerol kinase, thus consuming ATP rather than PEP. The haloarchaeal genomes do not encode any alternative route for the utilization of DHA (such as aldol condensation with glyceraldehyde-3phosphate to produce fructose-6-phosphate; Schurmann and Sprenger, 2001) suggesting that conversion of DHA to DHAP by either DHA kinase or glycerol kinase is the only available fate. The substrate promiscuity of glycerol kinase (Ouellette et al., 2013) may alleviate the need for a dedicated DHA kinase, particularly if the reaction is efficient enough to drive DHA uptake. Glycerol dehydratase (EC 4.2.1.30) Another avenue for glycerol utilization is conversion to 1,3propanediol (trimethylene glycol), and is used by bacteria during anaerobic growth on glycerol (Toraya et al., 1980; Forage and Lin, 1982; Seyfried et al., 1996; Raynaud et al., 2003). Glycerol is dehydrated by glycerol

dehydratase (or a broad-specificity diol dehydratase) to form 3-hydroxypropionaldehyde, which is reduced to 1,3propanediol by NADH-dependent 1,3-propanediol dehydrogenase (EC 1.1.1.202), thereby regenerating NAD1 for the fermentation of glycerol via the DHA pathway (Seyfried et al., 1996). This pathway has yet to be demonstrated for haloarchaea. For the haloarcheal genomes surveyed, candidate genes for glycerol dehydratase were identified only in the genome of Hac. jeotgali. Hac. jeotgali was isolated from fermented food (jeotgal) (Roh et al., 2007), suggesting a potential for anaerobic growth on glycerol and/or propanediol. However, it is not apparent from the sequence if this cobalamin-dependent enzyme is glycerol dehydratase or the related enzyme propanediol dehydratase (Bobik et al., 1997). In either case the Hac. jeotgali enzyme appears to have been recently acquired by horizontal gene transfer from bacteria (e.g., HacjB3_15946 [large subunit]: 67% match to Citrobacter freundii glycerol dehydratase large subunit; 64% match to Salmonella typhimurium propanediol dehydratase large subunit). In many bacteria, the glycerol/propanediol dehydratase gene cluster includes a putative transporter that is homologous to the putative haloarchaeal glycerol transporter mentioned above (see Glycerol uptake). Despite this association in bacteria, in Hac. jeotgali the gene for this putative transporter is located adjacent to the gene for glycerol kinase, not the glycerol/propanediol dehydratase genes. As such, in Hac. jeotgali the relationship between the dehydratase and the putative transporter, and the function of the dehydratase, is unclear. Concluding remarks: gene repertoire and haloarchaeal ecophysiology Of the 32 haloarchaeal genomes surveyed here, 27 show a genomic potential to utilize glycerol as a source of carbon and energy. These genomes encode glycerol kinase and at least one form of G3PDH (GlpABC or GlpA2). Genes for glycerol kinase, GlpABC-type G3PDH, and a putative glycerol transporter invariably form a gene cluster central to glycerol catabolism in haloarchaea, encoding a complete pathway for the conversion of glycerol to DHAP. Only in Hgm. borinquense and Hht. litchfieldiae is the GlpA2-type G3PDH also part of this gene cluster. In some species (Haloarcula hispanica, Haloarcula sp. CBA1115, Hht. litchfieldiae) this gene cluster also includes a gene for a putative chemotaxis methyl-accepting receptor protein, comprising an N-terminal methyl-accepting chemotaxis protein (MCP) signalling domain and a C-terminal domain that is homologous to the G3P-binding protein (UgpB) of the G3P ABC transporter (Fig. 2). Other species (Hfx. volcanii, Hfx. mediterranei, Hgm. borinquense) also encode this gene, but it is located elsewhere in the genome. This suggests a capacity to link the availability of G3P to swimming motility in these species, although this proposed


Glycerol metabolism of haloarchaea 873 chemotactic ability has yet to be experimentally demonstrated. All these species (like most of the haloarchaeal species surveyed) encode archaella (archaeal flagella) and hence possess the genomic potential for swimming motility. Overall, the G3P pathway for glycerol catabolism is more prevalent in haloarchaeal genomes than the alternative pathway via DHA. Moreover, genes for glycerol kinase and G3PDH encoding a complete pathway for glycerol conversion to DHAP (G3P pathway) are always found in the same genomes of glycerol-catabolizing haloarchaea. However, this is not true of the genes for glycerol dehydrogenase and DHA kinase (DHA pathway) as only four of nine genomes that encode glycerol dehydrogenase also encode DHA kinase (Fig. 2). There are several possible reasons for why the G3P pathway is favored over the DHA pathway, none of which are mutually exclusive. First, this pathway is initiated by phosphorylation of glycerol, thus preventing glycerol (converted to G3P) in the cytoplasm from diffusing out of the cell. Second, glycerol kinase is a high-affinity enzyme allowing the cell to utilize glycerol at low concentrations. Third, the G3P pathway is equipped to deal with both glycerol and G3P as substrates, and G3P is a component of cellular material. Finally, glycerol kinase can act on DHA (another Dunaliella byproduct) as well as glycerol, alleviating the requirement for a dedicated DHA kinase, providing DHA is available in the environment. The G3P pathway for glycerol catabolism to DHAP (Fig. 2) is present in all the species that have been reported to grow using glycerol as a growth substrate: Hbt. salinarum (Rawal et al., 1988), Hfx. volcanii (Kauri et al., 1990), Hfx. mediterranei (Garcia Lillo and Rodriguez-Valera 1990), Har. hispanica (Juez et al., 1986), Har. marismortui (Oren et al., 1988), Hrr. lacusprofundi (Franzmann et al., 1988), Halostagnicola larsenii (Castillo et al., 2006), Hgm. borinquense (Montalvo-Rodriguez et al., 1988), Hmc. mukohataei (Oren et al., 2002), Hqr. walsbyi (Bolhuis et al., 2004), and Hht. litchfieldiae (Williams et al., 2014). However, other species that encode this pathway have been reported not to grow on glycerol: Halopiger xanaduensis (Gutierrez et al., 2007), Hrd. utahensis (Wainø et al., 2000), Hrd. tiamatea (Antunes et al., 2008), Natrialba magadii (Tindall et al., 1984), and strain DL31 (Williams et al., 2014). The ability to utilize glycerol for growth is likely to be affected by the specific culture conditions employed. For example, in the study initially describing Hht. litchfieldiae it was reported that no growth occurred on glycerol (Mou et al., 2012). A subsequent study showed that when a Hht. litchfieldiae culture grown on medium containing both glycerol and pyruvate was transferred to medium containing glycerol as the sole defined carbon source, growth was observed (Williams et al., 2014). The strongest growth was observed when Hht. litchfieldiae was grown on both pyruvate and glycerol, suggesting that with the addition of pyruvate,

more DHAP can be directed to phospholipid synthesis and gluconeogenesis instead of the tricarboxylic acid cycle. The ability of some species to utilize glycerol while other species cannot appears to relate to specific abiotic characteristics of the hypersaline environments they inhabit, and/ or to an ability to perform niche adaptation. The species lacking identifiable genes for glycerol catabolism are: Haa. sulfurireducens, Halovivax ruber, Natronobacterium gregoryi, Nmn. pharaonis and Halobacterium sp. DL1. Haa. sulfurireducens is an obligate anaerobe found in anoxic environments (e.g., deep-sea salt-saturated anoxic Lake Medee; anoxic sediments and brines from hypersaline lakes in Kulunda Steppe, Russia) and is limited to growth using acetate and pyruvate as carbon sources, and elemental sulfur as the electron acceptor (Messina et al., 2016; Sorokin et al., 2016a,b). In soda lakes, instead of Dunaliella (Falb et al., 2008), haloalkaliphilic cyanobacteria are the major primary producers (Sorokin et al., 2014) and they synthesize glycine betaine, sucrose, trehalose, and glucosylglycerol as compatible solutes rather than glycerol €hn and Hagemann, 2011). The lack of glycerol catabo(Kla lism genes in Nbt. gregoryi and Nmn. pharaonis is consistent with both of them being found in soda lakes € per, 1982; Tindall et al., 1984). Interest(Soliman and Tru ingly, Natronococcus occultus was isolated from the same soda lake (Lake Magadi) as Nbt. gregoryi (Tindall et al., 1984) but possesses genes for glycerol catabolism, which may indicate that Ncc. occultus achieves niche adaptation by scavenging available glycerol (presumably low levels). Hvx. ruber and Hst. larsenii were both isolated from a hypersaline lake in Inner Mongolia, but the former cannot use glycerol as a growth substrate, whereas the latter can (Castillo et al., 2006; Castillo et al., 2007), which also indicates these species may colonize distinct trophic niches in the lake. During isolation of Hqr. walsbyi, this haloarchaeon only became prevalent at very low concentrations of glycerol (