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...

Comparative genomics reveals a common theme of 20S proteasome and proteasome-activating nucleotidase genes dispersed throughout archaeal genomes yet arranged in conserved linkages with gene homologues of translation and/or transcription machineries. To provide biological evidence for these linkages as well as insight into proteasome operon organization, transcripts of the five proteasomal genes of the halophilic archaeon Haloferax volcanii were analysed by Northern (RNA) blotting, RT-PCR and primer extension. These included psmA, psmB and psmC, encoding the 20S proteasomal subunits a1, b and a2, as well as panA and panB, encoding the PanA and PanB proteasome-activating nucleotidase proteins, respectively. All five of these genes are dispersed throughout the H. volcanii genome. For each proteasomal gene, a distinct transcript was detected by Northern blotting that was similar in size to the respective coding region. For both psmA and psmC, an additional transcript was detected that was 1.34 and 0.85 kb greater, respectively, than the coding region. Further analysis by Northern blotting and RT-PCR revealed that psmA was co-transcribed with genes encoding a Pop5 homologue of the RNase P endoRNase as well as an S-adenosylmethionine (SAM)-dependent methyltransferase. Likewise, psmC was co-transcribed with a downstream gene encoding a molybdenum cofactor sulfurase C-terminal (MOSC) domain protein. Additional proteasomal and neighbouring gene-specific transcriptional linkages were detected by RT-PCR. These results provide the first evidence that proteasome and tRNA modification genes are co-transcribed, reveal that a number of additional enzymes including those predicted to facilitate metal-sulfur cluster assembly are co-regulated with proteasomes at the transcriptional level, and provide further insight into proteasome gene transcription in archaea.