Background

Controlling the biofilm formation is crucial to understand the social interactions between bacteria in naturally occurring biofilm (Flemming et al., 2016). This is also particularly important for the biotechnological application of biofilms in biocatalysis, biosensing and waste treatment (Zhou et al., 2013; Jensen et al., 2016). The biofilm formation always begins with the bacterial adhesion to a substrate, which determines the spatial organization in biofilms (Liu et al., 2016; Nadell et al., 2016). Many strategies have been proposed to control bacterial adhesion such as modifying bacterial surface with bio-orthogonal reactive groups via liposome fusion (Elahipanah et al., 2016), immobilization of adhesive molecules on the substrates (Sankaran et al., 2015; Zhang et al., 2016; Peschke et al., 2017) and conjugating surface tags on bacteria (Poortinga et al., 2000; Rozhok et al., 2005; Lui et al., 2013). Among these, the light responsive approaches provide the highest spatiotemporal control, which is important to precisely control the fine structure of the biofilms. For instance, azobenzene linkers have been used as a photoswitchable tool to reversibly control the bacterial adhesion to substrates by altering the presentation of mannose, which is recognized by the bacterial surface receptor FimH (Voskuhl et al., 2014; Weber et al., 2014; Sankaran et al., 2015). In addition, azobenzene-based molecules have also been used to control bacteria adhesion to mammalian cells (Mockl et al., 2016), bacterial quorum sensing (Van der Berg et al., 2015) and biofilm formation (Hu et al., 2016) with UV-light. One of the major drawbacks of using UV-light is that it is toxic to bacteria. In this protocol, we present a new approach of how to control bacterial adhesion to substrates with blue light based on photoswitchable proteins. Besides being a non-invasive, reversible and tuneable technique to control bacterial adhesion to substrates, it also provides high spatiotemporal control required to form well-defined biofilms. Photoswitchable proteins are commonly used in the field of optogenetics to regulate gene expression, receptor activation and protein localization in cells with visible light (Müller and Weber, 2013; Tischer and Weiner, 2014). These optogenetic systems are very sensitive to visible light, bioorthogonal and noninvasive. Furthermore, these proteins are genetically encoded so they can be sustainably expressed in the cell. Here, we used the blue light responsive proteins, nMag and pMag, as photoswitches control bacterial adhesion. These proteins heterodimerize under blue light (480 nm) and dissociate from each other in the dark (Kawano et al., 2015). The strength and back conversion kinetics of the nMag and pMag interaction are different for the point mutants. The point mutant pMagHigh (and nMagHigh) has a stronger interaction with its binding partners and slower back conversion, while the opposite is true for the mutant pMagFast1 (and nMagFast1) (Zoltowski et al., 2009).

In our method we display the first interaction partner of the photoswitchable proteins, pMagHigh, pMag or pMagFast1 on the surface of E. coli using the circularly permutated OmpX (outer membrane protein X) protein (Daugherty, 2007). The pMag variants are attached through their C-terminal to the OmpX protein. The second interaction partner the photoswitchable protein, nMagHigh, is immobilized through a His6-tag at its C-terminal on a glass substrate with a PEG (polyethylene glycol) coating, which contains a Ni₂₊-NTA group (Schenk et al., 2014). This setup allows bacteria expressing pMag proteins on their surfaces to adhere to nMagHigh functionalized substrates under blue light when the two proteins interact but not in the dark. (Figure 1)


Figure 1. The engineered E. coli that express pMag proteins on their surface adhere to nMagHigh modified substrates under blue light. In the dark, the pMag-nMag interaction is reversed, which leads to the detachment of the bacteria from the substrate. Reproduced with permission from Chen and Wegner (2017).