Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius

Abstract

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes

“…The characterization of a series of null mutants defective in the glycogen metabolism pathway of Sulfolobus acidocaldarius DSM 639 has provided some insight. 285 Functional classification of enzymes can be achieved through glycogen content assays. Consequently, a mutation in either an anabolic or catabolic enzyme leads to a decrease or an excess of glycogen, respectively.…”

“…All glycogen phenotypes were in line with the presumed function of the catabolic glucoamylase-GH15 and anabolic glycogen synthase mutants, except for the null GH57amylase mutant, which displayed a dramatic decrease in glycogen level. 285 This atypical phenotype could be explained by the lack of branches within the storage polysaccharide, rendering it extremely sensitive to intracellular hydrolase activities. Interestingly, a similar phenotype was documented in the Arabidopsis branching enzyme mutant, wherein the chloroplast harbors multiple β-amylase activities that prevent the accumulation of linear glucan chains.…”

“…287 In line with this finding, a biochemical characterization of glgP null mutant of S. acidocaldarius displays a normal amount of glycogen in comparison to the wild-type strain, reinforcing the idea that GlgP is not a crucial enzyme in glycogen breakdown. 285 The designations GlgX and TreX were assigned to the debranching enzymes based on their presence in the glycogen or trehalose operons. Both enzymes are considered to be identical in terms of enzymatic properties.…”

“…The characterization of the null amylo-α(1→6) glucosidase mutant (GH133: AAY80544.1) of Sulfolobus, designated GlgX by the authors, results in an excess glycogen phenotype, suggesting its participation in the glycogen catabolism pathway. 285 In contrast to E. coli, archaea possess additional hydrolytic enzymes, including glucoamylase-GH15 and different glycosyl hydrolases GH57, which are controlled by catabolic inhibition. 290 These enzymes are secreted and have a function in starch utilization.…”

“…The characterization of a series of null mutants defective in the glycogen metabolism pathway of Sulfolobus acidocaldarius DSM 639 has provided some insight . Functional classification of enzymes can be achieved through glycogen content assays.…”