Construction and Characterization of tetH Overexpression and Knockout Strains of Acidithiobacillus ferrooxidans

Abstract

Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for bioleaching. It can obtain energy from the oxidation of Fe 2؉ , H 2 , S 0 , and various reduced inorganic sulfur compounds (RISCs). Tetrathionate is a key intermediate during RISC oxidation, hydrolyzed by tetrathionate hydrolase (TetH), and used as sole energy source. In this study, a tetH knockout (⌬tetH) mutant and a tetH overexpression strain were constructed and characterized. The tetH overexpression strain grew better on sulfur and tetrathionate and possessed a higher rate of tetrathionate utilization and TetH activity than the wild type. However, its cell yields on tetrathionate were much lower than those on sulfur. The ⌬tetH mutant could not grow on tetrathionate but could proliferate on sulfur with a lower cell yield than the wild type's, which indicated that tetrathionate hydrolysis is mediated only by TetH, encoded by tetH. The ⌬tetH mutant could survive in ferrous medium with an Fe 2؉ oxidation rate similar to that of the wild type. For the tetH overexpression strain, the rate was relatively higher than that of the wild type. The reverse transcription-quantitative PCR (qRT-PCR) results showed that tetH and doxD2 acted synergistically, and doxD2 was considered important in thiosulfate metabolism. Of the two sqr genes, AFE_0267 seemed to play as important a role in sulfide oxidation as AFE_1792. This study not only provides a substantial basis for studying the function of the tetH gene but also may serve as a model to clarify other candidate genes involved in sulfur oxidation in this organism.

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus

“…In order to measure growth or extract RNA, 1 mL of this culture was inoculated into 150 mL Starkey-S 0 or -K 2 S 4 O 6 liquid medium. Cell growth in Starkey-S 0 medium was measured at OD 600 after removal of elemental sulfur in the sample by low-speed centrifugation at 400 × g for 5 min (Yu et al, 2014). Only a small amount of cells (<1.5 %) were found attached to the sulfur particles, which were neglected in the cell growth measurement.…”

Multiple Osmotic Stress Responses in Acidihalobacter prosperus Result in Tolerance to Chloride Ions

“…Acidophilic bacteria have proven to be recalcitrant to the development of genetic methods, such as the creation of knockout mutants, and such techniques are only recently becoming more common (Wen et al, 2014; Yu et al, 2014). As a result, many acidophile studies have utilized “omics” techniques, including proteomics to investigate not only whole communities (Belnap et al, 2011; Mueller et al, 2011; Goltsman et al, 2013) but also specific responses in a single species (Baker-Austin et al, 2010; Mykytczuk et al, 2011; Potrykus et al, 2011; Mangold et al, 2013).…”

Metals and minerals as a biotechnology feedstock: engineering biomining microbiology for bioenergy applications

“…There has been a good deal of recent effort aimed at engineering biomining associated microbes [11][12][13][14][15][16][17][18][19][20][21][22][23] and understanding their unique physiology relevant for biomining operations, and more recently for developing new bioenergy applications [11,49,50].…”

“…Given that the dimensions of a bioleaching heap can be as large as one trillion liters, it is thought that metal biomining with chemolithoautotrophic bacteria represents the largest-scale bioprocess ever developed [10]. These processes rely on native microbial consortia, but reports on the engineering of these cells are beginning to emerge [11][12][13][14][15][16][17][18][19][20][21][22][23]. Future work aimed at the metabolic engineering of these cells for co-generation of useful chemicals and fuels using atmospheric CO 2 and the energy available in metal sulfide feedstocks could represent a new paradigm in bioproduction.…”