Sucrose Metabolism in Haloarchaea: Reassessment Using Genomics, Proteomics, and Metagenomics

Genomic and metagenomic surveys.(i) Sucrose uptake and hydrolysis. Sucrose uptake in haloarchaea does not appear to involve a PEP-PTS (7); sucrose PEP-PTS genes were not identified in the haloarchaeal genomes. Sucrose permease genes associated with sucrose uptake in bacteria (1) were also absent. The most likely mechanism for sucrose uptake involves ATP-binding cassette (ABC) transporters (CUT1 family) (22); relevant genes were found associated with those encoding glucose- or fructose-specific enzymes (especially glucose dehydrogenase or fructokinase, respectively). A gene for the enzyme responsible for sucrose hydrolysis was proposed in Har. marismortui (rrnAC1479) (14), and homologs (glycosyl hydrolase family 32) were found in a total of nine genomes (Fig. 3; see also Table S1 in the supplemental material).

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FIG 3

Genes related to sucrose metabolism encoded in haloarchaeal genomes. Data for utilization of sugars (sucrose [S], glucose [G], and fructose [F]) is based on previous reports (16, 18, 25, 26, 29, 33, 34, 62–76). Filled boxes indicate the presence of the gene(s), and colors are used to highlight related genes (e.g., genes from the same pathway); the absence of gene(s) is indicated by an x. GAPDH type I is associated with glycolysis whereas GAPDH type II is associated with gluconeogenesis, except in three species (Hst. larsenii, Htg. turkmenica, and Natrinema pellirubrum) in which GAPDH type II is assumed to operate in both directions (indicated by an exclamation point). ABC, ATP-binding cassette; ED, Entner-Doudoroff; FBP, fructose-1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFO, glucose-fructose oxidoreductase; GH15, glycoside hydrolase family 15; GH32, glycoside hydrolase family 32; KDG, 3-deoxy-2-oxo-d-gluconate; KDPG, 2-dehydro-3-deoxy-phosphogluconate; PEP, phosphoenolpyruvate; PEP-PTS, phosphoenolpyruvate-dependent phosphotransferase system; spED, semiphosphorylative ED pathway; XI/TIM, xylose isomerase-like, triose phosphate isomerase barrel domain. Har. vallismortis, Haloarcula vallismortis; Har. marismortui, Haloarcula marismortui; Har. hispanica, Haloarcula hispanica; Hfx. volcanii, Haloferax volcanii; Hfx. mediterranei, Haloferax mediterranei; Hmc. mukohataei, Halomicrobium mukohataei; Hht. litchfieldiae, Halohasta litchfieldiae; Hrr. lacusprofundi, Halorubrum lacusprofundi; Hgm. borinquense, Halogeometricum borinquense; Hst. larsenii, Halostagnicola larsenii; Nbt. gregoryi, Natronobacterium gregoryi; Htg. turkmenica, Haloterrigena turkmenica; Hrd. utahensis, Halorhabdus utahensis; Hrd. tiamatea, Halorhabdus tiamatea; Nnm. pellirubrum, Natrinema pellirubrum; Ncc. occultus, Natronococcus occultus; Hac. jeotgali, Halalkalicoccus jeotgali; Hpg. xanaduensis, Halopiger xanaduensis; Nab. magadii, Natrialba magadii; Hqr. walsbyi, Haloquadratum walsbyi; Hvx. ruber, Halovivax ruber; Nmn. pharaonis, Natronomonas pharaonis; Nmn. moolapensis, Natronomonas moolapensis; Hbt. salinarum, Halobacterium salinarum; Hbt. sp. DL1, Halobacterium sp. strain DL1; Haa. sulfurireducens, Halanaeroarchaeum sulfurireducens. DL31 is an undescribed Antarctic haloarchaeal genus (17).

The sucrose ABC transporter gene cluster often included two experimentally uncharacterized genes with protein domains suggestive of a role in glucose and/or fructose metabolism: COG0673 (predicted dehydrogenase; glucose-fructose oxidoreductase [GFO] domain) and COG1082 (sugar phosphate isomerase/epimerase; xylose isomerase-like, triosephosphate isomerase [XI/TIM] barrel domain) (Fig. 3, Table S1, and Fig. S1) (see also “(ii) Fructose catabolism” below in “Proteomics”). One or both genes were often located next to genes involved in sucrose catabolism, including fructokinase (Hht. litchfieldiae, Halostagnicola larsenii, Natronobacterium gregoryi, and Natronococcus occultus) (Fig. S1) and the putative sucrose ABC transporter (Hht. litchfieldiae, Hst. larsenii, Haloterrigena turkmenica, Ncc. occultus, and Natrialba magadii) (Fig. S1).

(ii) Haloarchaeal spED pathway. The essential enzymes for the degradation of glucose to pyruvate via the haloarchaeal spED pathway were encoded in most genomes of haloarchaeal species known to utilize sucrose and glucose: glucose dehydrogenase, gluconate dehydratase, KDG kinase, 2-dehydro-3-deoxy-phosphogluconate (KDPG) aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase (Fig. 1 and 3; Table S1). The last five enzymes listed comprise the lower shunt for the conversion of glyceraldehyde 3-phosphate to pyruvate that is common to both the ED and EMP pathways (including the modified versions of both in haloarchaea) and were present in all the surveyed haloarchaeal genomes (Fig. 3 and Table S1). There are two distinct and unrelated types of KDPG aldolases in haloarchaea: the bacterial-type (COG0800), and an archaeal-type (COG0329) (23). Both were represented among the surveyed haloarchaeal genomes, with some species encoding both types (Halorhabdus utahensis, Halorhabdus tiamatea, and Htg. turkmenica) and certain nonsaccharolytic species lacking both types (Natronomonas pharaonis, Halobacterium sp. strain DL1, and Halanaeroarchaeum sulfurireducens) (Fig. 3 and Table S1).

While the genes involved in the spED pathway were generally identifiable, there are a number of noteworthy clarifications. First, the immediate product of glucose dehydrogenase is gluconolactone, which can spontaneously hydrolyze to gluconate. However, this occurs slowly (especially at low temperatures), so a gluconolactonase may be required to catalyze this conversion (12). Lactonases can belong to multiple unrelated families and are therefore difficult to identify (24). The gluconolactonase provisionally identified in Haloferax volcanii (HVO_B0083) (15) does not have recognizable homologs in the majority of haloarchaeal genomes, including Antarctic Hht. litchfieldiae, which may be expected to have the gene as it grows in a cold environment. As such, the precise cellular mechanisms leading to hydrolysis of gluconolactone in the spED pathway remain to be determined.

Second, a glucose dehydrogenase gene is absent in the two Halorhabdus spp. even though they reportedly grow on glucose and fructose (but not sucrose) (25, 26). The two Halorhabdus sp. genomes were also unique among the 27 genomes in encoding a bacterial-like xylose isomerase (23), and in bacteria xylose isomerase functions in the interconversion of glucose and fructose (27). The growth properties of these species might be explained by fructose being converted to glucose and then entering the spED pathway via an unidentified glucose dehydrogenase or by glucose converted to fructose and catabolized via a pathway involving fructokinase (see “Haloarchaeal fructokinase” below).

Third, the inability of certain haloarchaea to metabolize glucose (Nmn. pharaonis, Halobacterium sp. DL1, and H. sulfurireducens) likely reflects the lack of an spED pathway (Fig. 3 and Table S1). However, despite encoding a complete spED pathway, some haloarchaea have been reported to lack the ability to utilize glucose as a carbon and energy source. This was observed for Halobacterium salinarum NRC-1 (28, 29) even though the enzyme activity of glucose dehydrogenase was found to be induced by glucose (30); this has been attributed to the absence of a catabolic GAPDH type I in Hbt. salinarum (31) (see the paragraph “Glucose catabolism and gluconeogenesis” in “Proteomics” below).

(iii) Haloarchaeal EMP pathway involving ketohexokinase. For fructose degradation, the haloarchaeal EMP pathway involves the same conversions as the classical EMP pathway, except that fructose is phosphorylated to fructose 1-phosphate rather than fructose 6-phosphate. In addition, fructose 1-phosphate is further phosphorylated to fructose 1,6-bisphosphate, which is an intermediate in the classical EMP pathway (12). The haloarchaeal EMP pathway for fructose catabolism is initiated by either ketohexokinase or fructose PEP-PTS. Cytoplasmic fructose is phosphorylated to fructose 1-phosphate in an ATP-dependent manner by ketohexokinase (7, 9), while fructose PEP-PTS generates fructose 1-phosphate during uptake of fructose (11) (Fig. 1).

The activity of haloarchaeal ketohexokinase has been characterized although the enzyme has not been identified (6–9). A candidate gene in Halomicrobium mukohataei has been proposed (23), and homologs are present in six genomes (Fig. 3 and Table S1), including the species with demonstrated ketohexokinase activity (Har. vallismortis, Har. marismortui, and Hfx. mediterranei) (6, 7, 9, 32). Pair-wise comparison of the experimentally derived, estimated amino acid composition of Har. vallismortis ketohexokinase (8) with the amino acid sequence of the Har. vallismortis candidate ketohexokinase showed a statistically significant positive correlation (r = 0.91) (Table S2), providing supporting evidence that they are the same protein. Overall, the above data support the hypothesis that the gene identified in Hmc. mukohataei (23) is responsible for producing ketohexokinase activity. The gene is present in six genomes that represent only three genera: Haloferax, Haloarcula, and Halomicrobium (Fig. 3 and Table S1). A total of nine of the 27 haloarchaeal genomes encode the components of a fructose PEP-PTS uptake system, including the three species with demonstrable ketohexokinase activity. Hmc. mukohataei is the only haloarchaeon surveyed here that encodes a putative ketohexokinase but lacks identifiable fructose PEP-PTS genes.

The enzyme that catalyzes the phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate is 1-phosphofructokinase (7, 9, 32). For Har. marismortui, 1-phosphofructokinase enzyme activity was shown to be very high for cultures growing on fructose (9). Twelve of the 27 genomes, including the majority of sugar-utilizing species, encode 1-phosphofructokinase (Fig. 3 and Table S1). All six species that encode ketohexokinase also encode 1-phosphofructokinase. Genes for 1-phosphofructokinase are present in six other species, four of which encode a fructose PEP-PTS (the Halorhabdus spp. do not) adjacent to the 1-phosphofructokinase gene (Fig. S1), suggesting that these species employ PEP-PTS and 1-phosphofructokinase for consecutive phosphorylations of fructose. A number of haloarchaea are reported to have the ability to grow on sucrose and fructose (18, 33, 34) but lack an identifiable 1-phosphofructokinase (Fig. 3 and Table S1), as has been noted for Halogeometricum borinquense (23). Collectively, these growth, enzymatic, and genomic data indicate that additional fructose degradation pathways are likely to exist in addition to the modified EMP pathway used by haloarchaea.

(iv) Haloarchaeal fructokinase. The genomes of 14 haloarchaeal species, including most sugar-utilizing haloarchaea, encode a gene annotated as fructokinase (COG0524). Haloarchaeal genomes that encode this putative fructokinase do not encode ketohexokinase and vice versa; the two genes are mutually exclusive among the surveyed genomes. Haloarchaeal fructokinases form a phylogenetic cluster with known fructokinases from bacteria and hyperthermophilic archaea (Fig. 4 and Fig. S2), providing circumstantial support for their functionality. A survey of metagenomes from hypersaline environments revealed that haloarchaeal fructokinase genes are far more abundant than haloarchaeal ketohexokinase genes: 1,467 versus 40 across 15 metagenomes representing five hypersaline habitats (Deep Lake, Antarctica; Cahuil Lagoon, Chile; Santa Pola, Spain; Isla Christina, Spain; Lake Tyrrell, Australia) (Table 1). Thus, despite the key role of ketohexokinase in the only characterized fructose degradation pathway in haloarchaea (12, 14, 23), ketohexokinase is not the dominant protein for fructose phosphorylation in haloarchaeal-dominated hypersaline environments.

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FIG 4

Maximum-likelihood phylogenetic tree of PFK-B sugar kinase family proteins. The tree includes fructokinase, KDG kinase, and 1-phosphofructokinase (84 proteins in total). Sulfofructokinase from Escherichia coli was used as the outgroup, and bootstrap values greater than 50% are reported. For the expanded tree, see Fig. S2 in the supplemental material.

Fructokinase catalyzes the ATP-dependent phosphorylation of fructose to fructose 6-phosphate (35–37). However, from genomic interrogation, a route by which fructose 6-phosphate could enter the EMP pathway in haloarchaea is not obvious. The enzyme 6-phosphofructokinase, which generates fructose 1,6-bisphosphate from fructose 6-phosphate, has not been identified in any haloarchaeal genome (14, 23, 28, 38) (Fig. 2b); this includes the classical ATP-dependent 6-phosphofructokinase, as well as ADP- and pyrophosphate-dependent archaeal 6-phosphofructokinases (14). One possibility is that fructose 6-phosphate could enter the EMP pathway using a novel haloarchaeal 6-phosphofructokinase, perhaps with broad specificity, as found for many phosphofructokinase B (PFK-B) family enzymes (39, 40). As a precedent in archaea, a PFK-B family phosphofructokinase/nucleoside kinase from Aeropyrum pernix exhibited a broad range of phosphoryl acceptors that included fructose 6-phosphate, fructose, and various nucleosides (40).

It has been proposed that fructose 6-phosphate may be converted to fructose 1-phosphate to enter the EMP pathway (23) (Fig. 2c); but a phosphofructomutase, an inducible enzyme in the bacterium Aeromonas hydrophila (41), has not been documented in archaea, and we did not identify obvious candidate genes in haloarchaeal genomes. In bacteria, fructose 6-phosphate can also be directed to the classical ED pathway (42) (Fig. 2d). However, again, there is no evidence that any haloarchaea have a complete classical ED pathway. Although haloarchaea encode the necessary enzymes for the conversion of fructose 6-phosphate to 6-phosphogluconate (glucose 6-phosphate isomerase and glucose 6-phosphate dehydrogenase) as part of the oxidative pentose phosphate pathway, haloarchaea lack an identifiable 6-phosphogluconate dehydratase gene for the conversion of 6-phosphogluconate to 2-keto-3-deoxy-d-gluconate (KDPG), and no 6-phosphogluconate dehydratase activity has been found in the haloarchaea for which this has been tested (Har. vallismortis and Hfx. mediterranei) (7). In the absence of an obvious path for fructose catabolism, we utilized proteomics to explore the possible fate(s) of fructose released from sucrose catabolism.

Proteomics.Hht. litchfieldiae was used as a model for haloarchaea that lack an identifiable ketohexokinase and can grow on sucrose and/or fructose. Forty-eight proteins were found to be differentially abundant (significantly higher or lower by 1.5-fold) in sucrose-grown cells (Table S3), out of a total of 1,096 detected proteins (Table S4). Fourteen of the differentially abundant proteins were noted for their potential relevance in sucrose metabolism (Fig. 5 and Table S3) and are discussed below.

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FIG 5

Possible sucrose metabolism pathways in Hht. litchfieldiae based on proteomic data. Proteins are indicated as follows: pink, sucrose-specific proteins involved in sucrose uptake and hydrolysis into glucose and fructose; blue, glucose-specific proteins associated with the semiphosphorylative ED pathway for glucose catabolism; orange, fructose-specific and oxidative pentose phosphate proteins; green, common semiphosphorylative ED pathway and EMP pathway proteins; red, gluconeogenesis proteins. Hypothetical reactions are indicated in gold. Enzymes that exhibited at least a 1.5-fold change in abundance in sucrose-containing medium have an arrow beside their names showing increased (upward arrow) or decreased (downward arrow) abundance. Note that of the two GFO domain proteins in the gene cluster, only halTADL_1905 was differentially abundant; halTADL_1905 and halTADL_1907 share 27% identity. ABC, ATP-binding cassette; DHAP, dihydroxyacetone phosphate; FBPase, fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFO, glucose-fructose oxidoreductase domain protein; KDG, 3-deoxy-2-oxo-d-gluconate; KDPG, 2-dehydro-3-deoxy-phosphogluconate; PEP, phosphoenolpyruvate; TrmB, putative TrmB transcriptional regulator; XI/TIM, xylose isomerase-like/triosephosphate isomerase barrel domain protein.

(i) Sucrose uptake and hydrolysis. Sucrose-containing cultures had higher abundances of the solute-binding lipoprotein (halTADL_1911) and ATPase (halTADL_1908) components of a CUT1 family ABC transporter (23), which we infer to be the sucrose importer in Hht. litchfieldiae (see “(i) Sucrose uptake and hydrolysis” in “Genomic and metagenomic surveys” above). The components are most closely related to characterized archaeal and bacterial carbohydrate import ABC transporters (43, 44), with sequence identities of 28% for the solute-binding lipoprotein and 45% to 46% for the ATPase.

Sucrose degradation may involve halTADL_0141 (glycosyl hydrolase family 15) because its abundance was higher in sucrose-grown cells. The homologs present in 14 other haloarchaea (Fig. 3 and Table S1) may play a similar functional role. Six of these genomes (not including Hht. litchfieldiae) also contained the β-fructofuranosidase (glycosyl hydrolase family 32) gene first proposed for sucrose hydrolysis in Har. marismortui (14) (Fig. 3 and Table S1). These hydrolase proteins lack signal peptides, which is consistent with cytoplasmic cleavage of sucrose (7). These data suggest that haloarchaea possess multiple mechanisms for sucrose hydrolysis, with some species possibly utilizing two different hydrolases.

(ii) Fructose catabolism. Glucose was detected in the supernatant of sucrose-grown Hht. litchfieldiae cultures soon after cultures began exponential growth; glucose concentrations (average of three replicates) were 28 µM, 52 µM, and 330 µM for 2, 4, and 6 days, respectively, after cultures entered exponential phase. In contrast, cultures grown only on pyruvate showed no glucose in the medium. The identification of a putative primary transporter and cytoplasmic hydrolase for sucrose in the proteome (see “(i) Sucrose uptake and hydrolysis” above) is consistent with nonphosphorylated glucose being leaked from cells into the medium rather than with extracellular sucrose breakdown.

However, the fate of fructose in Hht. litchfieldiae is unclear. Whereas the genomic and proteomic data point to a key role for fructokinase (halTADL_1913) in fructose metabolism, there is less evidence to describe how fructose 6-phosphate (the product of fructokinase) could enter glycolysis. It is possible that fructose 6-phosphate enters the oxidative pentose phosphate pathway. The requisite enzymes for the synthesis of ribulose 5-phosphate from fructose 6-phosphate were detected in the Hht. litchfieldiae proteome: glucose-6-phosphate isomerase (halTADL_0801), glucose-6-phosphate dehydrogenase (halTADL_0298), and 6-phosphogluconate dehydrogenase (halTADL_2122) (Table S4). Because we supplied both sucrose and pyruvate in the growth medium, it is possible that cells were able to direct fructose toward anabolic processes via the pentose phosphate pathway rather than to glycolysis (to make pyruvate). Thus, fructose could be fed directly into the pentose phosphate pathway using fructokinase, rather than indirectly via gluconeogenesis using fructose 1,6-bisphosphatase.

If fructose is used catabolically, it could possibly enter the spED pathway via an undescribed mechanism. In Har. marismortui, ¹³C-labeling studies showed that glucose was degraded exclusively via the spED pathway, whereas fructose was degraded predominantly via the haloarchaeal EMP pathway (96%), with a small fraction (4%) degraded via the spED pathway (9, 12). The Hht. litchfieldiae genome encodes two 1-phosphofructokinase proteins (Table S1), but neither was detected in the proteome. Although the absence of protein detection in proteomics does not rule out that the protein was synthesized by the cell, the nondetection of 1-phosphofructokinase is consistent with Hht. litchfieldiae not utilizing the haloarchaeal EMP pathway for degradation of fructose in the cytoplasm. Other proteins of the EMP pathway that were detected (Fig. 5 and Table S4) were not differentially abundant, and their presence is explained by the role they play in gluconeogenesis (see “(iii) Glucose catabolism and gluconeogenesis” below).

Fructose could potentially enter the spED pathway via a mechanism that involves the protein halTADL_1905 (COG0673; GFO domain protein) or the protein halTADL_1906 (COG1082; xylose isomerase-like, TIM barrel domain) (see “(i) Sucrose uptake and hydrolysis” above in “Genomic and metagenomic surveys”). While these proteins are not exclusively associated with haloarchaea possessing fructokinase (Fig. 3, Table S1, and Fig. S1), for Hht. litchfieldiae it is conspicuous that both proteins are encoded in the same gene cluster as the sucrose ABC transporter system and fructokinase, and both had higher abundances in sucrose-grown cells. In general, the COG0673 and COG1082 proteins are encoded in haloarchaeal genomes that also encode either fructokinase or ketohexokinase (Fig. 3 and Table S1), which suggests that they play roles in carbohydrate metabolism that are independent of fructose phosphorylation. The GFO domain of halTADL_1905 invites comparison with the enzyme GFO, which catalyzes the concomitant oxidation and reduction of glucose and fructose, respectively, to generate gluconolactone (an spED pathway intermediate) and sorbitol (glucitol) (45, 46) (Fig. 2e). However, we are doubtful that halTADL_1905 functions as a GFO. First, in the bacterium Zymomonas mobilis, which lives in sugar-rich environments, the sorbitol produced by periplasmic GFO is not metabolized but accumulates in the cytoplasm as an osmoprotectant in response to high extracellular sugar concentrations (45–47). Second, aside from Z. mobilis GFO, the GFO domain is present in other characterized enzymes, including a specific type of haloarchaeal xylose dehydrogenase (48) and the Bacillus subtilis glucose-6-phosphate 3-dehydrogenase (49). halTADL_1905 shows 25% to 27% sequence identity to the aforementioned enzymes and 27% identity to another GFO domain protein (halTADL_1907) in the same gene cluster as halTADL_1905 (Fig. 5) that was detected in the proteome but was not differentially abundant. Thus, GFO domains are found in diverse NAD(P)-binding enzymes associated with carbohydrate metabolism.

halTADL_1906 has a xylose isomerase-like, TIM barrel domain, which may mean that the enzyme catalyzes the isomerization of fructose to glucose; the glucose could enter the spED pathway (Fig. 2f), which would obviate a dedicated fructose degradation pathway involving fructokinase or ketohexokinase. Thus, both glucose and fructose moieties of sucrose could conceivably be degraded by the same pathway (spED pathway), separate from exogenous fructose (EMP pathway, initiated by concomitant uptake and phosphorylation by fructose PEP-PTS). In support of this possibility, a precedent for different glycolytic fates of endogenous and exogenous fructose is evident in certain bacteria. For example, in Rhodobacter capsulatus both the glucose and the fructose moieties of sucrose are catabolized via the classical ED pathway (35), whereas exogenously supplied fructose is imported by fructose PEP-PTS and degraded via fructose 1-phosphate and the classical EMP pathway (50).

(iii) Glucose catabolism and gluconeogenesis. Proteins involved in the breakdown of glucose to pyruvate via the spED pathway were detected in the Hht. litchfieldiae proteome, and the majority had higher abundances in sucrose-containing cells: glucose dehydrogenase (halTADL_0397), gluconate dehydratase (halTADL_0374), GAPDH type I (halTADL_0817), phosphoglycerate kinase (halTADL_0816), and pyruvate kinase (halTADL_3014) (Fig. 5 and Table S3).

All gluconeogenesis enzymes were also detected in the Hht. litchfieldiae proteome, including the irreversible enzymes fructose-1,6-bisphosphatase and PEP synthase (Table S4). Two PEP synthase homologs (halTADL_0698 and halTADL_1210) were among the most abundant proteins in cultures grown in sucrose-free medium compared to levels in sucrose-containing medium (Fig. 5 and Table S3), which accords with increased gluconeogenesis under these conditions. A carboxylate tripartite ATP-independent periplasmic (TRAP) transporter solute receptor of the TAXI family (halTADL_0243) (Table S3) also showed higher abundance in sucrose-free cultures, and we infer this protein to be involved in pyruvate uptake. The tricarboxylic acid (TCA) cycle proteins citrate synthase (halTADL_0686), aconitate hydratase (halTADL_2902), and succinate dehydrogenase SdhD subunit (halTADL_0444) showed lower abundances in sucrose-containing cultures (Table S3); this is consistent with a decreased contribution of the oxidative TCA cycle to energy conservation as a result of the increased generation of ATP and reducing equivalents through glycolysis.

The two GAPDH homologs showed contrasting abundances in response to carbon source: GAPDH type I (halTADL_0817) exhibited higher abundance in Hht. litchfieldiae grown in sucrose-containing medium, whereas GAPDH type II (halTADL_1211) exhibited lower abundance in these cultures; GAPDH type I and GAPDH type II showed the highest (increased 3.5-fold) and lowest (decreased 2.7-fold) abundances, respectively, of all detected proteins (Fig. 5 and Table S3). Similarly, in Hfx. volcanii growth on glucose resulted in higher expression of GAPDH type I but repression of GAPDH type II (12, 31, 51). This indicates inverse responses of the two GAPDH proteins, with GAPDH type I involved in glycolysis and GAPDH type II involved in gluconeogenesis (12, 31). An anabolic role for GAPDH type II is consistent with the absence of GAPDH type II in Halorhabdus, which lacks other identifiable genes for gluconeogenesis (23, 31). Most of the haloarchaea that possess GAPDH type II but lack GAPDH type I do not utilize carbohydrates and so would not be expected to perform glycolysis. However, three sugar-utilizing haloarchaeal species encode only GAPDH type II (no type I), which is presumably used in both directions (amphibolic) in these species, illustrating that dual GAPDH homologs (type I and type II) are not essential in all sugar-utilizing haloarchaea (Fig. 3 and Table S1).