Characterization of the iron-responsive transcription regulators in Pyrococcus furiosus
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Iron is an essential element for the growth of Pyrococcus furiosus since limited medium iron causes impaired growth. In previous iron-limitation microarray experiments, two putative iron transporters Ftr1 and FeoAB were significantly up-regulated in response to limited iron in the growth medium. This change in the iron acquisition system raises the question: what is responsible for the iron-dependent regulation? Two putative iron-responsive transcription factors were found in P. furiosus, Fur and DtxR. Studies on Fur-DNA binding activity and the characterization of its deletion mutant strain revealed no demonstrable role for the Fur homolog in the regulatory response of the cells to iron. Analysis of the DtxR genomic sequence suggested an incorrect translation start site prediction by NCBI and TIGR; another start site resulted in a 12 amino acid extension on the N-terminus. Recombinant DtxR proteins were expressed (with "full-length" and without the N-terminal extension) and their DNA-binding affinity was determined using electrophoretic mobility shift assay. The full-length DtxR was verified to bind specifically to ftr1 and feoAB promoters, indicating the function of DtxR as a potential transcriptional factor. The truncated DtxR failed to recognize any of the promoters. The dtxR deletion mutant (DTXR) did not show any growth phenotype compared to the control strain COM1C2 under iron-rich and iron-limited conditions. Microarray analysis of the iron-dependent regulation in DTXR and COM1C2 provided evidence that DtxR controls the expression of Ftr1 and FeoAB in P. furiosus since both iron transporters were no longer iron-regulated in the deletion strain. Results from the quantitative PCR experiment confirmed that DtxR is an iron-dependent repressor for the putative iron transporters in P. furiosus. Meanwhile, the ftr1 deletion mutant (FTR1) was constructed to investigate its role in iron uptake. However, no phenotype was observed for its growth in iron-limited and metal-limited medium; the similar intracellular iron content detected by ICP-MS in FTR1 and parent strain indicates that Ftr1 might not be an essential iron transporter despite its highly iron-responsive expression in P. furiosus.