Nitrogen metabolism of the haloarchaeon Haloferax volcanii

Cells in all domains of life have developed complex regulatory schemes to ensure nitrogen homeostasis. The mechanisms associated with nitrogen homeostasis have been examined in a wide variety of organisms and the molecular aspects of these regulatory systems present a view of an essential global regulatory program for each organism. Recognizing that a similar complex regulatory scheme was most likely present in the Archaea, we chose to investigate the response of Haloferax volcanii to variations in the amount and the quality of its nitrogen source as a model of global regulation in the Archaea.;Analysis of the recently sequenced H. volcanii genome showed that this organism encodes enzymes for the core ammonia assimilation pathways found in all organisms and several enzymes for the assimilation of nitrogen from alternative substrates. Phyletic distribution studies and phylogenetic analyses indicate that many of these alternative nitrogen assimilation pathways are absent in other Archaea and that they many have been acquired by the haloarchaea through horizontal gene transfer (HGT) from bacterial genomes. Inter-Domain HGT from Bacteria to the haloarchaea presents numerous barriers. Before such a gene product can be functionally assimilated, expression of the gene by the archaeal transcription machinery must be established, and in the haloarchaea, the gene product must have developed the necessary characteristics needed to function in the high salt cytoplasmic environment of these cells. Gene products, such as those involved in the catabolism of amino acids in the production of ammonium as a nitrogen source, also require that the expression of these genes be regulated to prevent unwanted degradation of these key building blocks in the cell.;To investigate the global transcriptome response of H. volcanii to changes in nitrogen availability, a genome-wide tiled array was constructed and used to characterize the RNA populations of cells undergoing balanced growth, during growth with a poor nitrogen source and under conditions of nitrogen starvation. Changes in the RNA populations indicated that genes encoding core nitrogen assimilation pathways showed differential expression similar to the patterns observed for the homologous pathways in Bacteria and other Archaea. However, the regulatory proteins common to the bacterial systems, and those described for Archaea, were absent in H. volcanii. An analysis of the RNAs identified a specific regulatory protein of the AsnC family in the negative regulation of the glnA gene encoding glutamine synthetase and showed that the general transcription factor (GTF) genes, tbp and tfb, also exhibited differential expression. Differential expression of the multiple GTF genes provides further support for the proposal that these proteins participate in directing expression of specific genes. These data also uncovered the regulated expression of numerous genes encoding uncharacterized proteins. A notable example was a gene encoding a serine/threonine protein kinase, prkA, which is present only in the haloarchaea. This gene exhibited high levels of RNA during nitrogen starvation, and other conditions of nutrient limitation, suggesting that this novel enzyme plays an important role in the physiological response to starvation in the haloarchaea.;Expression of the histidine utilization genes, hutUGIH, was examined in vivo as a model for the regulation of nitrogen assimilation genes that were acquired by HGT. In vivo studies established that the positive transcription regulator, HutR, regulated the hut operon genes when histidine or urocanate was provided to cells. The HutR protein is a member of the b&barbelow;acterio-o&barbelow;psin a&barbelow;ctivator (BOA) family of regulators and this class of regulator has not previously been associated with nitrogen regulation in any other organism. The results of this study provide a detailed view of the response of the haloarchaeon H. volcanii to nitrogen limitation and they have uncovered associations to genes of uncharacterized functions. These results have also established the specific regulatory mode of the hut genes, which have been acquired by HGT and are functionally assimilated into the metabolism of this haloarchaeon.