Insertion of Fluorescent Proteins Near the Plug Domain of MotB Generates Functional Stator Complexes

Many bacteria swim by the rotation of the bacterial flagellar motor (BFM). The BFM is powered by proton translocation across the inner membrane through the heteroheptameric MotA₅MotB₂ protein complex. Two periplasmic domains of MotB are critical in activating BFM rotation: (1) the peptidoglycan (PG) binding domain that anchors MotB in the PG layer and (2) the plug domain that modulates the proton flow. Existing cytoplasmic fluorescent probes have been shown to negatively affect motor rotation and switching. Here, we inserted a fluorescent probe in the periplasm near the plug of MotB to circumvent issues with cytoplasmic probes and for possible use in observing the mechanism of plug-based regulation of proton flow. We inserted green fluorescent protein and improved light-oxygen-voltage (LOV), a fluorescent version of the LOV domain, in four periplasmic locations in MotB. Insertions near the plug retained motility but showed limited fluorescence for both fluorophores. Additional short, flexible glycine-serine linkers improved motility but did not improve brightness. Further optimization is necessary to improve the fluorescence of these periplasmic probes.