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

Abstract

The γ-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.

Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper

“…Figure 1 shows the genomic contexts of all of these putative A. ferrooxidans genes. Based on their similarities to known genes, two ORFs encoding potential Cu P-type ATPases have been suggested (22). copA2 Af would be involved in Cu efflux, and copB Af in Cu import to the cytoplasm, as it has been proposed for copA from Enterococcus hirae (17).…”

“…ATPase (22) and showed a canonical metal-binding site ( Table 2). Although a downregulation of the expression of copB Af would be expected in the presence of a high concentration of Cu, copB Af was also induced by the presence of Cu during growth (Table 3).…”

Specificity of Fur Binding to the Oxidative Stress Response Gene Promoter in the Facultative Anaerobic Archaeon <i>Thermoplasma volcanium</i>

“…19) Several bacterial Fur deletion mutant strains are known to be sensitive to ROS. [20][21][22] These reports indicate that Fur is required for resistance to ROS. This study suggests the possibility that TvFur functions in the same way in Thermoplasma.…”

Draft Genome Sequence of the Extremely Acidophilic BacteriumAcidithiobacillus caldusATCC 51756 Reveals Metabolic Versatility in the GenusAcidithiobacillus

“…Of the three species, A. ferrooxidans is unique in also being able to obtain energy through the oxidation of ferrous iron, as well as being a facultative anaerobe capable of using ferric iron as an alternative electron acceptor. Acidithiobacilli have been shown to be able to fix atmospheric carbon via the Calvin-Benson-Bassham cycle (1,11,21) and to synthesize extracellular polymeric substances that are postulated to promote adhesion to mineral surfaces (3).As opposed to A. ferrooxidans, for which substantial bioinformatic and experimental evidence exists for these and other properties (4,14,19,20), A. caldus is poorly characterized, although it is known that it cannot carry out iron oxidation or nitrogen fixation (13). In contrast to the other two species of the genus, A. caldus thrives at temperatures up to 45 to 50°C.…”

“…As opposed to A. ferrooxidans, for which substantial bioinformatic and experimental evidence exists for these and other properties (4,14,19,20), A. caldus is poorly characterized, although it is known that it cannot carry out iron oxidation or nitrogen fixation (13). In contrast to the other two species of the genus, A. caldus thrives at temperatures up to 45 to 50°C.…”