Light-controlled bacteria-surface and bacteria-bacteria adhesions

Abstract

In bacterial biofilms, collective functions arise from the social interactions and spatial organization of bacteria. Controlling bacterial adhesion as key steps in biofilm formation with high spatial and temporal precision is essential for controlling the formation, organization and microstructure of biofilms. Light as a trigger provides unique advantages to dynamically manipulate bacterial interactions, including high spatiotemporal resolution and non-invasive, biocompatible and tunability remote control. In this thesis, different methods to control bacterial adhesions using light have been developed to control biofilm formation with high spatiotemporal precision and to study how the spatial organization of bacteria influences their collective functions. In chapter 2, I developed a method named bacterial photolithography that allows photopatterning biofilms with complicated geometries. In bacterial lithography, α-D-mannoside, which is recognized by the FimH surface receptor of Escherichia coli (E.coli), was linked to a non-adhesive poly(ethylene glycol) (PEG) surface through a photocleavable 2-nitrobenzyl linker. When a pattern of UV light in a desired shape was projected onto these surfaces, the light-exposed areas become non-adhesive and bacteria only adhered to the unexposed areas in the photopattern. This approach enabled bacterial patterning with high spatial resolution down to 10 µm without mechanical interference and the investigation of how microscale spatial organization affects collective bacterial interactions such as quorum sensing. In the following chapters photoswitchable proteins were used to control bacterial adhesions in an optogenetic approach. In particular, the protein pair nMag and pMag, which heterodimerizes under blue light and dissociates from each other in the dark were used as optogenetic building blocks. In chapter 3, I engineered bacteria to adhere specifically to substrates with high spatiotemporal control under blue light, but not in the dark, by using the nMag and pMag proteins as adhesins. For this, I expressed pMag proteins on the surface of E.coli so that these bacteria adhered to substrates with immobilized nMag protein under blue light. These adhesions were reversible in the dark and could be repeatedly turned on and off. Further, the number of bacteria that adhered to the substrate as well as their attachment and detachment kinetics were adjustable by using different point mutants of pMag and altering light intensities. Overall, this approach overcomes the problem of using UV light for photoregulation and chemically modifying the bacteria surface. Multi-bacterial communities are of fundamental importance and have great biotechnological potentials. However, controlling the assembly and organization of multicellular structures and therefore their function remains challenging. In chapter 4, I developed the first photoswitchable bacteria-bacteria adhesions and used these to regulate multicellularity and associated bacterial behavior. For this purpose, the proteins nMag and pMag were expressed on bacterial surfaces as adhesins. This allowed to trigger the assembly of multicellular clusters under blue light and reversibly disassemble them in the dark. These photoswitchable adhesions made it possible to regulate collective bacterial functions using light including aggregation, quorum sensing, biofilm formation and metabolic cross-feeding between auxotrophic bacteria. In a summary, light-responsive bacteria-surface and bacteria-bacteria adhesions allow controlling them with high spatiotemporal precision. While in a chemical approach bacterial lithography was used to pattern stable biofilms through irreversible light response, the photoswitchable proteins implemented in an optogenetic approach provide reversible and dynamic control over bacterial adhesions. All these tools open new possibilities for engineering multicellular communities, understand fundamental bacterial behavior in biofilms and design biofilms with new functions for biotechnological applications.