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 (⌬DTXR) 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 ⌬FTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ⌬FTR1 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 (⌬FUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus. T he hyperthermophilic anaerobic archaeon Pyrococcus furiosushas an optimal growth temperature of 100°C (1). It is of biotechnological interest as a source of highly thermostable enzymes and because of its ability to produce hydrogen gas (2, 3). It is well established that the organism requires iron for efficient growth, and a number of iron-containing enzymes involved in the primary metabolic pathways of P. furiosus have been characterized. These include three hydrogenases (MBH, SHI, and SHII) (4, 5), which are involved in hydrogen metabolism, superoxide reductase (SOR) and rubrerythrin, which are involved in the response to oxidative stress (6, 7), and the primary redox proteins, ferredoxin and rubredoxin, both of which contain iron (8, 9). Furthermore, ferritin and Dps of P. furiosus have been shown to be potential iron storage proteins, and the elemental-sulfur-induced protein A (SipA) was found to sequester intracellular iron and sulfide when iron and sulfur were present in the growth medium (10-12). Given the abundance of iron-containing proteins in this organism, the goal of the present study was to understand its response to iron availability.Iron metabolism is usually tightly regulated in aerobic organisms at the transcriptional level due to potential oxidative ...

Comparative Genomics of DtxR Family Regulons for Metal Homeostasis in Archaea

“…Both of these genes are preceded by the predicted FetR binding sites (see Tables S3 in the supplemental material). Specific binding of the PF0851 protein to the ftr1 and feoA promoter regions was further confirmed using EMSA (25). In Thermococcus kodakaraensis, the global transcriptional analysis of the TK0107 (FetR, clade 7) mutant has revealed 5-to 27-fold changes for the TK0652 (COG0428), TK0714-16 (feoAAB), and TK0958-57 (feoAB) operons under iron-limited conditions (26), confirming the predicted FetR-binding sites in the upstream regions of these three operons (see Table S3).…”

“…In A. fulgidus, electromobility shift assay (EMSA) and DNase footprinting have confirmed the binding ability of AF_1984 (MntR, clade 39) to its own promoter region (61), which is consistent with our data. In P. furiosus, the quantitative PCR analysis of the PF0851 (FetR, clade 7) deletion mutant has detected the differential expression of genes PF0723 (efeU or ftr1) and PF0858 (feoA) under iron-limiting conditions compared to the wild type (25). Both of these genes are preceded by the predicted FetR binding sites (see Tables S3 in the supplemental material).…”

“…The metalloregulators from the DtxR family also were implicated in the control of iron homeostasis in several archaeal species, including Halobacterium sp. strain NRC-1 and H. salinarium (23,24), Pyrococcus furiosus (25), and Thermococcus kodakaraensis (26).…”

Detecting differential growth of microbial populations with Gaussian process regression

“…SirR regulates metal uptake transporters at the level of transcription (Kaur et al 2006), repressing manganese uptake transporters under replete conditions. This regulatory link between metal homeostasis and oxidative stress is well-established in bacterial and eukaryotic organisms (Imlay 2003) but is only beginning to be appreciated in archaeal species (Zhu et al 2013). …”

Detecting differential growth of microbial populations with Gaussian process regression

“…Because metal excess 353 leads to oxidative stress through the Fenton reaction (Imlay 2003), this regulatory link between metal 354 homeostasis and oxidative stress is well-established in bacterial and eukaryotic organisms. However, this 355 connection in Archaea is only beginning to be appreciated (Zhu et al 2013).…”