Exploration of Retron Elements for the Genetic Engineering of <i>Acidithiobacillus ferrooxidans</i>

<i>Acidithiobacillus ferrooxidans</i> 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 <i>A. ferrooxidans</i> remains challenging due to limited molecular tools. In this study, we explored the feasibility of retron-based genetic engineering tools in <i>A. ferrooxidans</i> ATCC23270, leveraging a <i>pBAD</i> promoter system for inducible gene expression. We successfully expressed retron elements in <i>Escherichia coli</i> using the broad host pJRD215 plasmid and demonstrated both RNA and DNA antisense interference. We then expressed retron elements in <i>A. ferrooxidans</i> and targeted RNA antisense interference of the <i>PetA2</i> gene, which is a <i>bc</i>1 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 <i>A. ferrooxidans</i>. These findings establish retrons as a viable new tool for genetic modulation in <i>A. ferrooxidans</i><i>,</i> suggesting a new capability for metabolic engineering of acidophilic extremophiles.