Metabolic studies of the hyperthermophilic archaeon, Pyrococcus furiosus, using DNA microarrays
Schut, Gerrit Jan
MetadataShow full item record
This research developed a DNA microarray-based system for Pyrococcus furiosus and used this molecular tool to generate data to increase the understanding of the physiology of P. furiosus. Initially microarrays containing 271 ORFs were constructed and were used to study the differential regulation of these ORFs in cells grown in the presence and absence of elemental sulfur (Sº). Most of the ORFs whose expression was downregulated appeared to be related to the three multi-subunit hydrogenases in this organism, of which two are cytoplasmic and one is membrane bound. Down-regulation of the expression of these ORFs correlates well with the hydrogenase activities measured in cell-free extracts, indicating that none of these hydrogenases are directly involved in Sº metabolism. Two conserved hypothetical ORFs, now termed SipA and SipB (for sulfurinduced protein), were highly (>25-fold) up-regulated in Sº cultures and might participate in a novel Sº-reducing system. Using the methods developed by analyzing 271 ORFs, DNA microarrays were constructed for all 2065 ORFs annotated in the P. furiosus genome. These microarrays were used to analyze relative transcript levels in cells grown with peptide or with maltose as the primary carbon source. 126 ORFs showed differential expression levels of more than 5-fold between the two culture conditions and 82 of these appeared to be part of operons, indicating substantial coordinated regulation. Most of the ORFs up-regulated in maltose-grown cultures appear to be involved in amino acid metabolism, an incomplete citric acid cycle and glycolysis. Many of the ORFs upregulated in peptide cultures seem to be involved in the production of aryl and acyl acids from amino acids or encode enzymes involved in gluconeogenesis. The concentrations and types of aryl and acyl acids in spent growth media were consistent with the expression data. Interestingly, all ORFs encoding enzymes in the glycolysis and gluconeogenesis appeared to be monocistronic. The most strongly regulated were those encoding the enzymes glyceraldehyde-3-P dehydrogenase, phosphoglycerate kinase and fructose-1,6-bisphosphatase, suggesting that the glycolytic and gluconeogenic pathways in the P. furiosus are very tightly regulated.