Project description:Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases.
Project description:The outer-membrane c-type cytochrome Cyc2 is generally considered to be the initial electron acceptor in iron respiratory chain of Acidithiobacillus ferrooxidans ATCC 23270, a model microorganism in acidophilic bioleaching environment. In our work, however, the knockout of cyc2 did not result in impaired Fe(II) consumption or growth capacity. To screen the potential genes for alternative initial electron acceptors other than Cyc2, RNA-Seq was employed to compare global gene expressions in the A. ferrooxidans ATCC 23270 wild type and the Δcyc2 mutant grown on Fe(II) or switched energy source from S0 to Fe(II). The data focused on 29 up-regulated and 19 down-regulated genes in the mutant under both conditions, among which AFE_1428 was the most highest one. in-silico analysis also suggested that the product of AFE_1428 might act as an alternative initial electron acceptor when Cyc2 was absent, which needs to be further validated.
Project description:Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.
Project description:Bioleaching processes or microbially mediated iron/sulfur redox processes in acid mine drainage (AMD) result in mineral dissolution and transformation, the release of mercury and other heavy metal ions, and changes in the occurrence forms and concentration of mercury. However, pertinent studies on these processes are scarce. Therefore, in this work, the Fe/S redox-coupled mercury transformation mediated by Acidithiobacillus ferrooxidans ATCC 23270 under aerobic and/or anaerobic conditions was studied by combining analyses of solution behavior (pH, redox potential, and Fe/S/Hg ion concentrations), the surface morphology and elemental composition of the solid substrate residue, the Fe/S/Hg speciation transformation, and bacterial transcriptomics. It was found that: (1) the presence of Hg2+ significantly inhibited the apparent iron/sulfur redox process; (2) the addition of Hg2+ caused a significant change in the composition of bacterial surface compounds and elements such as C, N, S, and Fe; (3) Hg mainly occurred in the form of Hg0, HgS, and HgSO4 in the solid substrate residues; and (4) the expression of mercury-resistant genes was higher in earlier stages of growth than in the later stages of growth. The results indicate that the addition of Hg2+ significantly affected the iron/sulfur redox process mediated by A. ferrooxidans ATCC 23270 under aerobic, anaerobic, and coupled aerobic-anaerobic conditions, which further promoted Hg transformation. This work is of great significance for the treatment and remediation of mercury pollution in heavy metal-polluted areas.
Project description:Cadmium is one of several heavy metals present in contaminated soils. Apparently, it has no biological role but can produce DNA damage, overexpression of stress response proteins and misfolded proteins, amongst other deleterial effects. Acidithiobacillus ferrooxidans is an acidophilic bacterium capable of resisting very high concentrations of heavy metals such as cadmium. This is important for industrial bioleaching processes where Cd+2 concentrations can be in the range of 5-100 mM. Cadmium resistance mechanisms in these microorganisms have not been fully characterized. A. ferrooxidans ATCC 53993 contains genes coding for possible metal resistance determinants such as efflux systems belonging to three families: P-type ATPases, RND transporters and cation diffusion facilitators (CDF). In addition, it has some extra copies of these genes in its exclusive genomic island (GI). Several of these putative genes were characterized in the present report by determining their transcriptional expression profiles and functionality. Moreover, a global quantitative proteomic analysis was carried out to further explore new cadmium resistance determinants in this biomining acidophile. Changes in iron oxidation pathways, upregulation of transport proteins (P-type ATPases and CDFs) and changes in ribosomal protein levels were seen. Finally, increased concentrations of exclusive putative cadmium ATPases present in strain ATCC 53993 GI and other non-identified proteins such as Lferr_0210, which forms part of a possible operon, could explain its greater resistance to cadmium compared to other acidophiles such as A. ferrooxidans ATCC 23270.