Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus

Abstract

Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

FIG. 1.

Agarose gel electrophoresis of the cDNA amplicon (Amp) and the first (DP1) and second (DP2) difference products from the mRDA of ferrous iron- versus sulfur-grown S. metallicus. Tester cDNA was from (a) ferrous iron-grown cells and (b) sulfur-grown cells. M, 100-bp ladder.

FIG. 2.

A comparison of ORFs in genome fragments of S. metallicus (ORFs of >300 bp) or S. tokodaii. S. tokodaii ORFs and corresponding fox genes are connected by diagonal lines, and the S. tokodaii gene numbers are given below the S. metallicus gene names.

FIG. 3.

Relative expression levels of ORFs of the sequenced S. metallicus genome fragment and of the sulfur oxygenase-reductase gene (sor) assessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures. Mean values and standard deviations are from analyses of triplicate cultures grown autotrophically on ferrous iron, pyrite, or sulfur.

FIG. 4.

Investigation of the transcriptional organization of S. metallicus ORFs using RT-PCR with primers situated in adjacent ORFs. Products produced from cDNAs of a ferrous iron-grown culture (Fe) or a pyrite-grown culture (P) or from genomic DNA (G) are shown.

FIG. 5.

Coomassie blue-stained 2D blue native/SDS-PAGE gels of membrane preparations of Sulfolobus metallicus cells grown on pyrite (a) and sulfur (b). The putative FoxA is indicated as one of three spots (circled) that represent a possible protein complex present in pyrite-grown cells but not sulfur-grown cells. The positions of molecular mass markers (in kDa) are shown on the borders of the gels.