Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile Acidithiobacillus ferrooxidans

Abstract

The gamma-proteobacterium Acidithiobacillus ferrooxidans lives in extremely acidic conditions (pH 2) and, unlike most organisms, is confronted with an abundant supply of soluble iron. It is also unusual in that it oxidizes iron as an energy source. Consequently, it faces the challenging dual problems of (i) maintaining intracellular iron homeostasis when confronted with extremely high environmental loads of iron and (ii) of regulating the use of iron both as an energy source and as a metabolic micronutrient. A combined bioinformatic and experimental approach was undertaken to identify Fur regulatory sites in the genome of A. ferrooxidans and to gain insight into the constitution of its Fur regulon. Fur regulatory targets associated with a variety of cellular functions including metal trafficking (e.g. feoPABC, tdr, tonBexbBD, copB, cdf), utilization (e.g. fdx, nif), transcriptional regulation (e.g. phoB, irr, iscR) and redox balance (grx, trx, gst) were identified. Selected predicted Fur regulatory sites were confirmed by FURTA, EMSA and in vitro transcription analyses. This study provides the first model for a Fur-binding site consensus sequence in an acidophilic iron-oxidizing microorganism and lays the foundation for future studies aimed at deepening our understanding of the regulatory networks that control iron uptake, homeostasis and oxidation in extreme acidophiles.

Figure 1.

Pipeline for the computational and experimental steps used to identify candidate Fur-binding sites in A. ferrooxidans. Additional information and results can be found in Supplementary Data S1.

Figure 2.

Predicted Fur-regulated gene clusters and associated predicted Fur boxes grouped into four main functional categories: iron acquisition, iron utilization, transporters and transcriptional regulators. Arrows representing each gene indicate direction of transcription and are not drawn to scale. The double-hashed line separating independent gene clusters indicates that they are not contiguous in the genome.

Figure 3.

EMSAs of ³²P-labeled DNA fragments (probes) containing predicted Fur boxes and promoters of (A) gloA and feoP, (B) copB and abcS4, (C) iscR and phoB, (D) hppH and (E) fdx1 in the presence of Fur_AF (Fur), Fur_AF plus anti-Fur serum (α-Fur) or cold probe (P*) as indicated. (F) EMSA of a ³²P-labeled probe containing no predicted Fur box and corresponding to a low G + C DNA fragment of pUC18 vector. 1 = probe DNA, (A–D) 2 = shift with 300 nM Fur_AF, 3 = supershift with anti-Fur antibody; (E) 2 = shift with 300 nM Fur_AF, 3 = shift with 400 nM Fur_AF, 4 = supershift, 5 = cold probe competition, 6 = absence of shift in the presence of the anti-Fur antibody alone. (F) absence of shift in the presence of: 2 = 100 nM Fur_AF, 3 = 200 nM Fur_AF, 4 = 300 nM Fur_AF, 5 = 400 nM Fur_AF and occurrence of shift in the presence of 6 = 500 nM Fur_AF.

Figure 4.

In vitro transcription assays (IVTAS) of genes with predicted Fur boxes. (A) Scheme of each gene showing the sequence and relative position of the predicted Fur box with respect to cognate promoters. (B) IVTAs in the presence (+) or absence (−) of 300 nM Fur_AF and 100 µM Mn(II). Primers used for IVTAs are listed in Table 2. M = molecular standards in bps. Arrow heads indicate where a transcript is absent after the addition of Fur_AF and Mn(II).

Figure 5.

DNA sequence logos of A. ferrooxidans Fur-binding sites derived from several bioinformatic and experimental prediction strategies. (A) E. coli Fur box consensus sequence (3), (B) Heterologous training set logo (66 Fur boxes, Supplementary Data S1A), (C) A. ferrooxidans information-theory-based logo (90 Fur boxes, Supplementary Data S1B), (D) A. ferrooxidans HMM-based logo (79 Fur boxes, Supplementary Data S1D), (E) A. ferrooxidans Fur box logo derived for EMSA-validated genes (9 Fur boxes), (F) A. ferrooxidans consensus Fur-binding sequence. Boxed and blue letters represent bases that are protected by Fur DNA binding in E. coli (31,32,54).