Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon: Pyrococcus furiosus Grown on Carbohydrates or Peptides

Abstract

The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of Pyrococcus furiosus (optimal growth temperature, 100°C). This was used to determine relative transcript levels in cells grown at 95°C with either peptides or a carbohydrate (maltose) used as the primary carbon source. Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition. Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation. Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins. A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products. A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation. Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides. In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold-three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis. The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data. The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that P. furiosus can readily adapt to a change in its primary carbon source.Hyperthermophiles are microorganisms that grow optimally at temperatures of 80°C and above (53). Most are classified as archaea, and many utilize peptides as a carbon source and reduce elemental sulfur (S 0 ) to H 2 S (53). Some, including several species of Pyrococcus and Thermococcus, are able to metabolize poly-and oligosaccharides as well as peptides (5,6,16). Herein, we focus on the primary carbon metabolism of one of the best studied of all hyperthermophiles, Pyrococcus furiosus. This organism was isolated from a shallow marine volcanic vent and grows optimally near 100°C with either peptides or carbohydrates used as its carbon and energy sources (16).Prior studies of the primary pathways for carbon metabolism by P. furiosus have yielded several surprises. In particular, it contains a modified Embden-Meyerhof pathway with two ADP-rather than ATP-dependent kinases, glucokinase and phosphofructokinase (26,55). In addition, the expected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the phosphoglycerate kinase (...

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

“…At the level of GAP oxidation, three alternative enzymes have been characterized: the ubiquitous GAPDH/PGK couple, and the non-phosphorylating archaeal counterparts GAPOR and GAPN (Table 2). Detailed enzymatic characterization as well as transcription analysis in P. furiosus and T. tenax (Brunner et al 1998Lorentzen et al 2004;Schäfer and Schönheit 1993;Schut et al 2003;Van der Oost et al 1998) whose genomes encode all three GAP converting enzymes (Table 2) revealed a catabolic role for GAPN and GAPOR and an anabolic role for the GAPDH/ PGK couple. These findings have been confirmed by a recent, excellent mutational approach in the hyperthermophilic anaerobe Thermococcus kodakarensis, which harbors all three GAP converting enzymes (Matsubara et al 2006).…”

“…The latter two enzymes catalyze the unidirectional direct oxidation of GAP forming 3-phosphoglycerate but differ in their co-substrate specificity [pyridine nucleotides (GAPN), ferredoxin (GAPOR)] (Brunner et al 1998;Mukund and Adams 1995;Siebers and Schönheit 2005;Van der Oost et al 1998). Biochemical studies and transcriptional data in T. tenax and Pyrococcus furiosus, which harbor all three GAP converting enzymes, revealed that GAPN and GAPOR substitute for the anabolic enzyme couple NADP + -dependent GAPDH and phosphoglycerate kinase (PGK) in these hyperthermophiles at the expense of substrate-level phosphorylation (Brunner et al 1998Lorentzen et al 2004;Schäfer and Schönheit 1993;Schut et al 2003;Van der Oost et al 1998). In S. solfataricus only two GAP converting enzymes, classical GAPDH and GAPN, were identified (Verhees et al 2003).…”

Insights into ABC Transport in Archaea

“…This would argue against a bacterial-like regulation system of the maltose system and would make the C-terminal extension of MalK obsolete. However, DNA microarray studies in P. furiosus, which contains the identical genomic region as T. litoralis (DiRuggiero et al, 2000), showed that all the genes are upregulated upon growth on maltose while trmB and malK stay upregulated even upon growth on peptide sources (Schut et al, 2003). This suggests that MalK also in the archaeal system is involved in the regulation of the expression of the mal operon, although this hypothesis awaits further experimental proof.…”

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

“…Although the P. furiosus FNOR, like SfrAB, also catalyses NADPH-dependent benzyl viologen reduction (Ma & Adams, 1994, 2001Schut et al, 2003), the amount of NADPH-dependent benzyl viologen reductase activity in the soluble fraction of the acetate-adapted SfrAB-null strain was less than 1 % of the activity present in the wild-type strain. The apparent lack of FNOR activity in the adapted mutant despite a three-to sixfold increase in the level of its putative transcripts could have several possible explanations, including insufficient expression despite elevated mRNA levels, post-transcriptional regulation, or differences in the activity, stability or substrate specificity of the putative G. sulfurreducens FNOR relative to that of P. furiosus.…”

“…GSU3057 and GSU3058 were previously annotated as a homotetrameric NADPHdependent glutamate synthase and a putative dihydroorotate electron transfer subunit, respectively . However, no NADPH-dependent glutamate synthase activity could be detected in either wild-type or acetateadapted SfrAB-null extracts, and GSU3057 and GSU3058 were found to be 65.6 % and 65.2 % similar, respectively, to the a and b subunits of the NADPH-dependent ferredoxin oxidoreductase (FNOR) of Pyrococcus furiosus (Ma & Adams, 1994, 2001Schut et al, 2003). Phylogenetic analysis of GSU3057 and related proteins, including SfrB, which is 36 % similar, clearly indicated that GSU3057 was more closely related to the a subunit of P. furiosus FNOR than to the small subunits of the characterized glutamate synthases (Fig.…”