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

Abstract

The canonical pathway for sucrose metabolism in haloarchaea utilizes a modified Embden-Meyerhof-Parnas pathway (EMP), in which ketohexokinase and 1-phosphofructokinase phosphorylate fructose released from sucrose hydrolysis. However, our survey of haloarchaeal genomes determined that ketohexokinase and 1-phosphofructokinase genes were not present in all species known to utilize fructose and sucrose, thereby indicating that alternative mechanisms exist for fructose metabolism. A fructokinase gene was identified in the majority of fructose- and sucrose-utilizing species, whereas only a small number possessed a ketohexokinase gene. Analysis of a range of hypersaline metagenomes revealed that haloarchaeal fructokinase genes were far more abundant (37 times) than haloarchaeal ketohexokinase genes. We used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase) and identified changes in protein abundance that relate to growth on sucrose. Proteins inferred to be involved in sucrose metabolism included fructokinase, a carbohydrate primary transporter, a putative sucrose hydrolase, and two uncharacterized carbohydrate-related proteins encoded in the same gene cluster as fructokinase and the transporter. Homologs of these proteins were present in the genomes of all haloarchaea that use sugars for growth. Enzymes involved in the semiphosphorylative Entner-Doudoroff pathway also had higher abundances in sucrose-grown H. litchfieldiae cells, consistent with this pathway functioning in the catabolism of the glucose moiety of sucrose. The study revises the current understanding of fundamental pathways for sugar utilization in haloarchaea and proposes alternatives to the modified EMP pathway used by haloarchaea for sucrose and fructose utilization.IMPORTANCE Our ability to infer the function that microorganisms perform in the environment is predicated on assumptions about metabolic capacity. When genomic or metagenomic data are used, metabolic capacity is inferred from genetic potential. Here, we investigate the pathways by which haloarchaea utilize sucrose. The canonical haloarchaeal pathway for fructose metabolism involving ketohexokinase occurs only in a small proportion of haloarchaeal genomes and is underrepresented in metagenomes. Instead, fructokinase genes are present in the majority of genomes/metagenomes. In addition to genomic and metagenomic analyses, we used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase but lacks ketohexokinase) and identified changes in protein abundance that related to growth on sucrose. In this way, we identified novel proteins implicated in sucrose metabolism in haloarchaea, comprising a transporter and various catabolic enzymes (including proteins that are annotated as hypothetical).

Keywords: archaea.

FIG 1

Known sucrose catabolism pathways in haloarchaea. The pathways show the separate fates of glucose degraded by the semiphosphorylative ED pathway and fructose degraded by the modified EMP pathway: purple, sucrose-specific steps; blue, spED pathway; red, modified EMP pathway; green, common shunt. Note that the conversion of glucose to gluconate in the spED pathway involves two steps: oxidation of glucose to gluconolactone, followed by spontaneous hydrolysis, or hydrolysis catalyzed by an unidentified gluconolactonase, to gluconate. 1-PFK, 1-phosphofructokinase; ABC, ATP-binding cassette; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KDG, 3-deoxy-2-oxo-d-gluconate; KDPG, 2-dehydro-3-deoxy-phosphogluconate; PEP, phosphoenolpyruvate; PEP-PTS, PEP-dependent phosphotransferase system; S-layer, surface layer.

FIG 2

Alternative fructose catabolism pathways. The pathways shown include those ruled out for Hht. litchfieldiae based on genomic interrogation and proteomic data (indicated by a red cross). (a) Modified EMP pathway, known only for certain haloarchaea. The absence of an identifiable ketohexokinase precludes this pathway in the majority of fructose-utilizing haloarchaea. (b) Classical EMP pathway. The absence of an identifiable 6-phosphofructokinase precludes this pathway in fructose-utilizing haloarchaea. (c) Classical EMP pathway with phosphofructomutase, an enzyme not known in haloarchaea. (d) Classical ED pathway. The absence of an identifiable 6-phosphogluconate dehydratase precludes this pathway in fructose-utilizing haloarchaea. (e) Novel hypothetical pathway which entails the concomitant oxidation and reduction of glucose and fructose, providing an intermediate (gluconate) that can enter the haloarchaeal semiphosphorylative ED pathway. (f) Novel hypothetical pathway which entails the conversion of fructose to glucose, which can enter the spED pathway. Novel enzymes proposed for the pathways in panels e and f are shown in gold. Note that, for simplicity, the conversion of glucose to gluconate is represented by a single arrow. DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFO, glucose-fructose oxidoreductase; KDG, 3-deoxy-2-oxo-d-gluconate; KDPG, 2-dehydro-3-deoxy-phosphogluconate; PEP, phosphoenolpyruvate; XI/TIM, xylose isomerase-like, triosephosphate isomerase barrel.

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).

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.

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.