DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus: evidence for anNew type of sulfur-reducing enzyme complex

Abstract

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

FIG. 1

Fluorescence intensities of DNA microarrays. (A) cDNA versus cDNA derived from the same cultures of cells grown on maltose (no S°). (B) cDNA versus cDNA derived from cells grown on maltose with or without S°. The upper and lower diagonal lines indicate fivefold changes in the signal intensities. See the text for details.

FIG. 2

Expression of ORFs encoding hydrogenase-related subunits with or without S°. The ORFs encoding the subunits of the cytoplasmic hydrogenase I (A), cytoplasmic hydrogenase II (B), and the membrane-bound hydrogenase (C) are arranged according to their positions in their respective operons. These are plotted against signal intensities for cDNA obtained from cells grown with (solid bars) or without (shaded bars) S°. The results with all subunits have a confidence level of at least 96% (P > 0.98 for the cytoplasmic hydrogenases and P > 0.96 for the membrane-bound enzyme).

FIG. 3

Genome organization of sipA and sipB. The back to back ORFs coding for SipA (179 amino acids) and SipB (115 amino acids) are shown with the indicated directions for transcription. Putative transcription stop sites are indicated with a “ball on a stick,” and putative translational start sites are indicated with right-angled arrows. The putative start sites are preceded with possible ribosomal binding sites (underlined) and TATA boxes (boxed). The start site for sipB shown here is not the same as that given in the genome annotation (which is indicated with an asterisk [54]). Both sipA and sipB contain an inverted repeat (I-1 and I-2) directly next to the proposed TATA boxes. The position of the ORF (1873389) encoding the SipB extension (142 amino acids) is indicated.

FIG. 4

Expression of the ORFs encoding POR and VOR with or without S°. The genes (A, B, and D) encoding the α-, β-, and δ-subunits of POR and of VOR are arranged according to their positions in their respective operons, along with the gene (G) encoding the γ-subunit that is shared by the two enzymes (31). For each ORF, the signal intensities are indicated for cDNA obtained from cells grown with (solid bars) or without (shaded bars) S°.