Characterization of glycerol metabolism and related metabolic pathways in the haloarchaeaon Haloferax volcanii

The molecular mechanisms surrounding carbon utilization and its regulation are not well characterized in haloarchaea. Glycerol is a readily-abundant energy source for halophilic, heterotrophic communities as a result of its large-scale production by the halotolerant green algae Dunaliella sp. This study sought to characterize glycerol catabolism in the model haloarchaeon Haloferax volcanii. This work provides evidence that glycerol is a preferred carbon source over glucose and that the former is metabolized through chromosomally-encoded glycerol kinase ( glpK) and glycerol-3-phosphate dehydrogenase (gpdA1B1C1). Both glpK and gpdA1 transcripts were glycerol-inducible, and the enzymatic activity of their gene products was not inhibited by glucose. Furthermore, glpK and gpdA1B1C1 are under the control of a strong, glycerol-inducible promoter. The glycerol metabolic operon also includes a putative glycerol facilitator as well as a homolog of the bacterial phosphotransferase system (PTS) protein Hpr. Additional bacterial PTS homologs EI and EII are encoded in the H. volcanii genome and preliminary evidence suggests that these EI and EIIBFru homologs may be involved in H. volcanii fructose metabolism. This work also examines the regulation of haloarchaeal carbon metabolism in H. volcanii, including the characterization of a DeoR/GlpR-type transcriptional repressor of glucose and fructose metabolic enzymes. The putative DeoR/GlpR-type protein encoded by glpR is transcriptionally-linked to pfkB encoding phosphofructokinase (PFK). Based on qRT-PCR, enzyme activity, and transcriptional reporter analyses, GlpR is likely a transcriptional repressor of genes encoding PFK and 2-keto-3-deoxygluconate kinase, two key enzymes of haloarchaeal fructose and glucose metabolism, respectively. This GlpR protein purified as a tetramer by gel filtration chromatography under both high and low salt and is postulated to use a phosphorylated intermediate of either glucose and/or fructose metabolism as its ligand. A putative repressor binding motif consisting of an inverted hexameric repeat was identified adjacent to characterized archaeal promoter consensus motifs upstream of the kdgK1 and glpR-pfkB genes. Taken together, our results provide insight into the carbon metabolic pathways of various investigated heterotrophic haloarchaea.