Identification and characterization of quorum-quenching enzyme AiiF from <i>Acidithiobacillus ferrooxidans</i> and its application for biofilm disruption

ABSTRACT Quorum sensing-induced functions, including biofilm formation, can greatly influence biotechnology applications, including industrial metal bioleaching processes. Biofilm formation by the acidophilic chemolithoautotrophs Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans is a key factor affecting mineral attachment and leaching efficiency, and is mediated by N -acyl homoserine lactone (AHL) quorum-sensing systems. Here, we identified an AHL lactonase gene native to A. ferrooxidans strains TFBk and BY-3, termed AiiF, which suppresses AHL-dependent quorum sensing. The kinetic parameters of AHL degradation by AiiF recombinantly expressed in Escherichia coli were obtained and represent a relatively slow reaction ( K m of 3.5 ± 2.2 mM and k cat of 0.15 ± 0.04 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mstyle> <mml:mrow> <mml:mstyle displaystyle="false"> <mml:msup> <mml:mi mathvariant="normal">s</mml:mi> <mml:mrow> <mml:mo>−</mml:mo> <mml:mn>1</mml:mn> </mml:mrow> </mml:msup> </mml:mstyle> </mml:mrow> </mml:mstyle> </mml:math> ), where the high K m value is similar to its closest relative, AiiA. AHL docking simulations and in vivo biosensor data suggest that AiiF is mechanistically promiscuous and exhibits broad substrate specificity. Exogenous addition of AiiF disrupted biofilm formation in laboratory strains of both A. ferrooxidans and A. thiooxidans . Overexpression of recombinant AiiF in A. thiooxidans demonstrated that quorum quenching can reduce biofilm formation and influence growth behavior on a sulfur substrate. IMPORTANCE Biomining of critical materials with metal sulfide-oxidizing bacteria can reduce costs and environmental impacts. The relationship between biofilm formation and bioleaching efficiency is complex, with different mineral systems being enhanced or inhibited by biofilm formation. We identified and characterized a new lactonase enzyme native to Acidithiobacillus ferrooxidans , AiiF, that can degrade N- acyl homoserine lactone (AHL) autoinducers. This enzyme can reduce biofilm formation in bioleaching microbes, and this activity may be leveraged to enhance future biomining activities.