Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon Haloferax volcanii

Abstract

Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8-and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.Halophilic and halotolerant microorganisms have adapted different methods for withstanding the high osmotic pressure exerted by their surrounding hypersaline environment. Halophilic archaea (7, 13), as well as the halophilic bacterium Salinibacter ruber (19), maintain a high intracellular salt concentration by accumulating K ϩ and Cl Ϫ ions and excluding Na ϩ ions, thus requiring intracellular proteins to be active under high-salt conditions. Many halophilic bacteria (24), the halotolerant green alga Dunaliella sp. (3), and some haloarchaea (12) exclude cytoplasmic salts and rely on organic solutes such as ectoine, glycine betaine, and glycerol to provide osmotic balance. Glycerol, in particular, is accumulated in molar quantities by Dunaliella as an organic osmotic solute. Due to leakage from healthy Dunaliella cells (1, 3, 26) and/or cellular lysis, glycerol is released into the surrounding environment, where it serves as a primary energy source for haloarchaea. Upon uptake, halophilic microorganisms assimilate glycerol into dihydroxyacetone phosphate (DHAP) by one of two catabolic routes (Fig. 1). In one route, glycerol is first phosphorylated by glycerol kinase to form sn-glycerol-3-phosphate, which is subsequently oxidized by sn-glycerol-3-phosphate dehy...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…volcanii deletion mutants of glycerol kinase (⌬glpK) as well as glycerol phosphate dehydrogenase (⌬glpA1) were incapable of growth on glycerol. In the presence of glycerol, glycerol-mediated catabolite repression of glucose metabolism was more pronounced in the ⌬glpK mutant than in the wild-type strain (557).…”

“…In Hfx. volcanii, glycerol-mediated catabolite repression of glucose metabolism has been reported (557). Glycerol is a preferred carbon source for haloarchaea, because it is highly abundant in high-salt environments.…”

“…Glycerol is accumulated as a compatible solute in the halotolerant green alga Dunaliella to protect the cell from osmotic stress and is released into the environment by leakage and cell lysis (558). Biochemical and genetic studies revealed that glycerol is channeled into the central carbohydrate metabolism via glycerol kinase (glpK; HVO_1541) and the glycerol phosphate dehydrogenase complex (glpA1B1C1; chromosomal) (556,557). Hfx.…”

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

“…Fluorescence and growth of these cell cultures Quantitative real-time PCR (qRT-PCR). Total RNA was isolated from 1.5 ml of cells (OD 600 0.7) using the RNeasy Mini kit for bacteria (Qiagen) as previously reported , with modifications as previously described (Sherwood et al, 2009). Total RNA concentration was measured by A 260 and quality was determined by 0.8 % (w/v) agarose gel electrophoresis in 16 TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0) after heating the RNA samples to 50 uC for 15 min.…”

“…Total RNA concentration was measured by A 260 and quality was determined by 0.8 % (w/v) agarose gel electrophoresis in 16 TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0) after heating the RNA samples to 50 uC for 15 min. qRT-PCR was performed on total RNA preparations as previously described (Sherwood et al, 2009) using the iQ SYBR Green Supermix and iCycler MyiQ real-time PCR detection system (Bio-Rad) with primers specific for smrs-gfp and ribL10 (ribosomal protein). The ribL10 primers (RibL-RT_f and RibL-RT_r) were as reported elsewhere and served as a normalization control (Brenneis et al, 2007).…”