Exploration of Retron Elements for the Genetic Engineering of Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophilic bacterium widely found in industrial biomining operations. The cells obtain energy through iron and/or sulfur oxidation, and the resulting ferric iron can solubilize copper and other critical materials. The genetic engineering of acidophiles including A. ferrooxidans remains challenging due to limited molecular tools. In this study, we explored the feasibility of retron-based genetic engineering tools in A. ferrooxidans ATCC23270, leveraging a pBAD promoter system for inducible gene expression. We successfully expressed retron elements in Escherichia coli using the broad host pJRD215 plasmid and demonstrated both RNA and DNA antisense interference. We then expressed retron elements in A. ferrooxidans and targeted RNA antisense interference of the PetA2 gene, which is a bc1 complex gene involved in the sulfur metabolism electron transport chain. Engineered strains exhibited reduced expression of sulfur oxidation genes and increased iron oxidation under high sulfur conditions, demonstrating retron-mediated regulation. While transcriptional interference was evident, genome editing in concert with the addition of a single-stranded annealing protein (SSAP), or recombineering, was not detected in A. ferrooxidans. These findings establish retrons as a viable new tool for genetic modulation in A. ferrooxidans, suggesting a new capability for metabolic engineering of acidophilic extremophiles.