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 ofHalohasta 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-grownH. litchfieldiaecells, 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.IMPORTANCEOur 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 ofHalohasta 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).

Open Issues for Protein Function Assignment in Haloferax volcanii and Other Halophilic Archaea

“…However, the activity was not assigned to a gene. Detailed bioinformatic analyses have been made [ 308 , 309 ] and point to a small set of orthologs represented by Hmuk_2662, the ortholog of HVO_1812 (for further details, see Supplementary Text S1 Section S9 ).…”

“…(b) The assignment of fructokinase activity to the Hht. litchfieldiae candidate gene halTADL_1913 (UniProt:A0A1H6QYL4) is based on a differential proteomic analysis [ 309 ] (see Supplementary Text S1 Section S9 for details). Very close homologs are rare in haloarchaea.…”

Open issues for protein function assignment inHaloferax volcaniiand other halophilic archaea

“…However, the activity was not assigned to a gene. Detailed bioinformatic analyses have been made (Anderson et al, 2011, Williams et al, 2019 and point to a small set of orthologs represented by Hmuk_2662, the ortholog of HVO_1812 (for further details see Suppl. Text S1 Section 9).…”

“…(b) The assignment of fructokinase activity to the Hht. litchfieldiae candidate gene halTADL_1913 (UniProt:A0A1H6QYL4) is based on differential proteomic analysis (Williams et al, 2019) (see Suppl. Text S1 Section 9 for details).…”

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase

“…The Archaean dominance is even more clear in extreme environments such as salt lakes [ 10 ] and acid lakes [ 11 ], where the microbial diversity is limited through the extreme environments and the low overall abundancy of nutrients. In many of these cases, carbon inputs for these Archaea [ 12 ] are provided only by a few photosynthetically active organisms (cyanobacteria, algae) [ 13 , 14 ]. One can imagine that symbiotic interactions or associations become even more critical to establish stable microbial communities in such environments like, for instance, the hypersaline Tuz Lake in inland Turkey [ 15 , 16 ].…”