(599ab) Display of Rogfp on Cell Surface Enables in Situ Quantification of Extracellular Redox Status in Biofilms

In natural and engineered habitats, microorganisms often exist as surface-associated microbial communities known as biofilms, where the cells are embedded within a matrix of self-produced extracellular polymeric substances (EPS) including polysaccharides, proteins, nucleic acids and lipids. The microenvironments in biofilms are highly dynamic and heterogeneous with spatiotemporally varying concentration of nutrients, chemical cues, and signaling molecules because of the interplay between biofilm metabolism and physicochemical processes in biofilm matrix. Previous studies have suggested an important role of various physicochemical processes in biofilm development. Among these processes, redox changes influence a wide range of cellular respiration processes. Redox reactions are often associated with the biological processes involved with the release or retention of energy. It has been reported that the redox status is highly critical for the microorganisms to cope with environmental stresses. The redox state has also been demonstrated to have a great impact in driving the morphological development of biofilms. To understand the role of redox state in biofilm development and responses of biofilms to environmental perturbations, it is essential to quantify the redox state of microenvironments in biofilms. Microsensors have been widely used to quantify with spatial resolution physicochemical parameters such as pH, dissolved oxygen, redox potential, and metabolites in biofilms. In addition, certain redox sensitive dyes can be introduced into biofilms to map redox state of microenvironments. However, these methods might perturb the biofilm structure, as they are locally invasive.

Genetically encoded redox biosensors offer a non-invasive alternative approach to quantify the redox state in real time within biofilms. Redox sensitive green fluorescent protein (roGFP) is one such redox biosensor that monitors the redox status through the ratiometric quantification of relative fluorescence intensity at two excitation maxima (400 & 480 nm). The roGFP probe is constructed by the formation of disulfide bonds between the cysteine residues on the surface of GFP β-barrel. The disulfide formation during the oxidation of roGFP increases the excitation peak at 400 nm relative to 480 nm excitation peak. Similarly when the roGFP is in its reduced state, a higher excitation peak is exhibited at 480 nm. Therefore, the redox state can be monitored by measuring the ratio between the fluorescence intensity at 400 nm and 480 nm excitation maxima. The intracellularly expressed roGFP has been reported to be effective to monitor  cellular redox state in the presence of membrane permeable oxidants like hydrogen peroxide (H₂O₂) or reductant like dithiothreitol (DTT). However, the roGFP redox probe has never been exploited to quantify redox state in extracellular microenvironments of biofilms. 

The goal of this study was to develop an approach for quantifying redox state in extracellular microenvironments of biofilms through displaying roGFP at the cell exterior. Specifically, we used Shewanella oneidensis, a bacterium of great environmental and biotechnological interest, as a model organism and genetically fuse roGFP (~ 27 kDa) onto the C-terminus of BpfA, a large protein (~285 kDa) mainly expressed on cell surface and in biofilm matrix.  Then we grew biofilms using this reporter strain and demonstrated the quantification of depth-resolved redox state in the extracellular microenvironments of the biofilms.