@article {40227,
	title = {Biofilm engineering through c-di-GMP tuning boosts bioleaching efficiency and arsenic tolerance in Acidithiobacillus ferrooxidans},
	journal = {Applied and environmental microbiology},
	year = {2026},
	pages = {17},
	abstract = {Bioleaching offers a sustainable alternative to conventional metallurgy, but its application is limited by low leaching rates, inhibition by heavy metals, and prolonged adaptation. Here, we engineered Acidithiobacillus ferrooxidans, a model bioleaching microorganism ubiquitous in mining environments, by modulating intracellular bis(3' -5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling to enhance biofilm formation, bioleaching efficiency, and arsenic tolerance. Overexpression of diguanylate cyclase genes AFE_1379, AFE_0053, and AFE_1373 produced engineered strains S-222, S-306, and S-651, respectively, with 1.7-, 2.5-, and 5-fold higher intracellular c-di-GMP levels than the control carrying the empty plasmid vector. Under arsenic-free condi tions, all engineered strains showed similar growth profiles, but S-306, at intermediate c-di-GMP (306.3 {\textpm} 28.1 μg mg-1), formed cytochrome-rich biofilms with low internal resistance and achieved the highest bioleaching efficiency. Under arsenic stress, S-651, at high c-di-GMP (651.4 {\textpm} 15.5 μg mg-1), developed polysaccharide-rich biofilms that enhanced arsenic tolerance, scorodite (FeAsO$_{4}${\textperiodcentered}2H$_{2}$O) precipitation, and bioleaching performance. Transcriptomic analysis confirmed these strain-specific gene expression patterns. These findings demonstrate that tuning intracellular c-di-GMP enables A. ferrooxidans to reprogram biofilm matrix composition for extracellular electron uptake and heavy-metal resistance, providing a synthetic biology strategy for environmentally friendly bioleaching and tailings recycling},
	url = {https://journals.asm.org/doi/full/10.1128/aem.02288-25},
	author = {Han, Xi and Yidan Hu and Yanbo Yue and Yuefei Ding and Bin Cao and Shi, Liang and Juan Liu}
}
