Characterization of endogenous promoters for control of recombinant gene expression in Acidithiobacillus ferrooxidans

Abstract

Acidithiobacillus ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes, which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. Acidithiobacillus ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here, we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism.

Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

“…The sfGFP operon and the KDC operon were inserted into pBAM2 to generate the pBAM2-GFP and pBAM2-KDC plasmids (Fig. S2) (6,12). The plasmids were electroporated into donor E. coli S17-1 pir lacI cells for conjugation.…”

“…While the use of mercury resistance has been explored as an alternative selectable marker, heavy metals are undesirable for handling in cultures because of their toxicity (11). It has recently been reported that robust growth of engineered A. ferrooxidans cells was possible in liquid media containing ferric iron and sulfur, indicating that ferrous iron is not necessary for A. ferrooxidans cultivation (12). Therefore, it was hypothesized that selection procedures may be improved with the removal of ferrous iron from the solid and liquid selection media.…”

“…The wild-type (WT) strain and the genetically modified strains have been investigated using a variety of medium compositions, including the addition of metal chelators, to improve growth rates and maximize chemical yields in batch and continuous cultures (16,18,19). Although several constitutive promoters of different strengths have been characterized with respect to driving gene expression, the need for inducible control of gene expression within this organism led to the identification of endogenous promoter sequences compatible with the growth conditions for A. ferrooxidans (12,20). Thus, these developments with plasmid-based systems have greatly expanded the ability to modify A. ferrooxidans cells for different future applications.…”

Harnessing a methane‐fueled, sediment‐free mixed microbial community for utilization of distributed sources of natural gas

“…In addition to building a symbiosis with ANME to enable methane consumption, sulfate‐reducing bacteria can remediate heavy metals (García, Moreno, Ballester, Blázquez, & González, ; Joo, Choi, Kim, Kim, & Oh, ) and produce plastic precursor storage molecules (Hai, Lange, Rabus, & Steinbüchel, ; Wang, Yin, & Chen, ). Sulfide generated through this process can be used as feedstock for genetically tractable chemoautotrophs (Kernan, West, & Banta, ; Nybo, Khan, Woolston, & Curtis, ), which can make high‐value chemical products (Kernan et al, ). Finding a conductive physical scaffold to enable industrially‐relevant reactions within the context of an ecophysiologicaly sustainable system—whereby microbial communities can take advantage of natural electrical and chemical gradients (e.g., Pfeffer et al, )—would decrease transport and scale‐up costs while building a robust, customizable system.…”

“…| 1451 Rabus, & Steinbüchel, 2004;Wang, Yin, & Chen, 2014). Sulfide generated through this process can be used as feedstock for genetically tractable chemoautotrophs (Kernan, West, & Banta, 2017;Nybo, Khan, Woolston, & Curtis, 2015), which can make high-value chemical products (Kernan et al, 2016). Finding a conductive physical scaffold to enable industrially-relevant reactions within the context of an ecophysiologicaly sustainable systemwhereby microbial communities can take advantage of natural electrical and chemical gradients (e.g., Pfeffer et al, 2012)-would decrease transport and scale-up costs while building a robust, customizable system.…”