Microfluidics based exploration for quorum quenching genes in Antarctic microbiomes

Quorum sensing (QS) is a form of microbial communication that enables each cell in a population to sense the total population density, so that gene expression can be modified accordingly. Quorum quenching (QQ) is the antagonistic disruption of this communication by competing organisms, with potential use in the ongoing human effort to control microbial populations. Previous studies have taken advantage of functional metagenomics to retrieve new QS/QQ genes, but the frequency of obtaining positive clones remained very low, suggesting the need for increased screening efficiency. Here, a new ultrahigh-throughput screening method was developed to search for genes encoding novel QQ genes based on functional metagenomics, microfluidics, and the development of an Escherichia coli reporter strain whose fluorescence is repressed in the presence of AHLs but restored by the expression of any gene that interferes with the QS system. This strain was transformed with a metagenomic short-insert library collected from Antarctic plant rhizospheres; an understudied extreme environment. The library was encapsulated in droplets containing single clones and sorted using fluorescence-activated cell sorting (FACS). After screening approximately 7,000,000 clones, around 200 were recovered and one positive hit was confirmed, showing a previously unreported mode of action that would have been difficult to detect using rational computational methods. These findings underline the potential of microfluidics to dramatically increase screening efficiency, while reducing costs and processing time, and they act as a proof of concept for the discovery of more genes involved in QS and other molecular mechanisms of interest in microbial ecology. IMPORTANCEQuorum sensing (QS) regulates essential microbial behaviours, including virulence, biofilm formation, and symbiotic interactions, making it a key target for ecological and applied microbiology. Disrupting QS through quorum quenching (QQ) has major potential in agriculture, medicine, and biotechnology as an alternative to antibiotics or chemical treatments. However, the discovery of QQ genes through brute-force approaches is limited by large screening efforts, resulting in high costs and long processing times. Our study introduces a microfluidics-based, ultrahigh-throughput functional screening platform that strongly improves screening efficiency. It allowed us to identify a novel QQ gene, predicted to be involved in a previously undescribed mechanism of QS disruption, from an under-explored extreme environment (Antarctic rhizosphere). These results demonstrate how microfluidics can optimise screening technologies, while unlocking the hidden functional diversity of microbial communities useful for biotechnological applications in microbial control.