Regulation of iron metabolism by Pyrococcus furiosus

Abstract

Iron is an essential element for the hyperthermophilic archaeon Pyrococcus furiosus, and many of its iron-containing enzymes have been characterized. How iron assimilation is regulated, however, is unknown. The genome sequence contains genes encoding two putative iron-responsive transcription factors, DtxR and Fur. Global transcriptional profiles of the dtxR deletion mutant (ΔDTXR) and the parent strain under iron-sufficient and iron-limited conditions indicated that DtxR represses the expression of the genes encoding two putative iron transporters, Ftr1 and FeoAB, under iron-sufficient conditions. Under iron limitation, DtxR represses expression of the gene encoding the iron-containing enzyme aldehyde ferredoxin oxidoreductase and a putative ABC-type transporter. Analysis of the dtxR gene sequence indicated an incorrectly predicted translation start site, and the corrected full-length DtxR protein, in contrast to the truncated version, specifically bound to the promoters of ftr1 and feoAB, confirming its role as a transcription regulator. Expression of the gene encoding Ftr1 was dramatically upregulated by iron limitation, but no phenotype was observed for the ΔFTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ΔFTR1 and the parent strain were similar, suggesting that under the conditions tested, Ftr1 is not an essential iron transporter despite its response to iron. In contrast to DtxR, the Fur protein appears not to be a functional regulator in P. furiosus, since it did not bind to the promoters of any of the iron-regulated genes and the deletion mutant (ΔFUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus.

Fig 1

Characterization of the ΔDTXR strain. (a) Growth of ΔDTXR and the parent (COM1c2) strains under iron-sufficient (+Fe, 10 μM iron added) and iron-limited (−Fe, no iron added) conditions. Growth was monitored by optical density at 660 nm. Results are shown for COM1c2 (circles) and ΔDTXR (triangles) under iron-sufficient (solid symbols) and iron-limited (open symbols) conditions. The arrow indicates the culture harvest point for RNA isolation for DNA microarray analysis. (b) The effect of dtxR deletion on gene expression was measured in ΔDTXR and COM1c2 using quantitative PCR. Total RNA was prepared from ΔDTXR and COM1c2 grown under iron-sufficient and iron-limited conditions. The constitutively expressed gene encoding the pyruvate ferredoxin oxidoreductase (POR) gamma subunit (PF0971) was used as an internal control. Results are expressed as a change in gene expression, comparing ΔDTXR to COM1c2 under iron-sufficient (closed bars) and iron-limited (open bars) conditions.

Fig 2

Characterization of the ΔFTR1 mutant. (a) Growth of ΔFTR1 and COM1 under iron-sufficient [+Fe, 10 μM (NH₄)₂Fe(SO₄)₂ added] and iron-limited (−Fe, no iron source added) conditions. Cultures were grown in 125-ml bottles at 98°C with 5 g/liter maltose, 0.5 g/liter yeast extract, and 20 μM uracil. Cell growth was monitored by assaying total cell protein at each time point. Results are shown for COM1 (squares) and ΔFTR1 (circles) under iron-sufficient (closed symbols) and iron-limited (open symbols) conditions. (b) Cultures of ΔFTR1 (open bars) and COM1 (closed bars) grown under iron-limited (Lim) and iron-sufficient (Suf) medium were harvested at exponential (LOG) and stationary (STAT) phases. Cell pellets were washed three times with 1× base salt solution to remove extracellular metals and lysed using lysis buffer. Lysates were then centrifuged, and the supernatant was collected for the measurement of intracellular iron content using ICP-MS. The iron contents measured by ICP-MS were normalized to the protein concentration of each sample.