Glycerol-mediated repression of glucose metabolism and glycerol kinase as the sole route of glycerol catabolism in the haloarchaeon Haloferax volcanii.
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.
Sherwood
Katherine E
KE
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700, USA.
Cano
David José
DJ
Maupin-Furlow
Julie A
JA
D002851:Chromatography, High Pressure Liquid; D019641:DNA, Archaeal / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal / Q000235:genetics; D020745:Genome, Archaeal / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D005991:Glycerol Kinase / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D019641:DNA, Archaeal; D005993:Glycerolphosphate Dehydrogenase; D005991:Glycerol Kinase; D005947:Glucose; D005990:Glycerol