Engineering sticky microbes: A framework for appraisal of microbial cell-cell adhesion

Engineering bacterial consortia is key in biotechnological applications where specific spatial interactions among their components determine system performance. Genetic tools for programming cell-cell binding such as the nanobody-antigen (Nb-Ag; relying on non-covalent binding) and SpyCatcher-SpyTag (Spy; based on covalent linkage) systems are increasingly adopted as assets for enabling specific interactions of interest among otherwise detached members of a given combination of strains or species. Yet, despite the potential of these technologies, a robust metrology to quantify and compare the efficacy, stability, and specificity of inter-cell binding in a consortium is lacking. This work introduces a conceptual and experimental framework to systematically evaluate synthetic binding systems by combining aggregation assays, flow cytometry, and microscopy. Together, these techniques provide detailed insights into the structure and composition of engineered bacterial aggregates. This methodology was benchmarked with two Escherichia coli strains tagged with different fluorescent markers and engineered to surface-display matching adhesins. Under the test conditions, results showed that the Spy system outperformed the Nb-Ag system in terms of aggregation speed and stability. We also applied the same metrics for assessing the efficacy of the Spy system to mediate synthetic consortia of the pathogenic bacterium Enterobacter cloacae. The robustness of the results suggests the proposed framework to become a valuable tool for quantitative studies on designer bacterial consortia.