Constitutive, endogenous, fluorescent membrane reporters for dynamic cell cycle analysis in Bacillus subtilis

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis that, when fused with a fluorophore, would cause the membrane to fluoresce in a constitutive, uniform, and bright manner. A forward genetic transposon-based approach, combined with fluorescence-activated cell sorting, was used to identify a fluorescent fusion to the glucose PTS transmembrane transport protein PtsG with all desired properties. Moreover, PtsGΩmsfGFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsGΩmsfGFP to track cell growth in microfluidic channels and modify the definition of cytokinesis as when membrane fluorescence reached a local maximum at the division plane, a definition further supported by fluorescence loss in photobleaching (FLIP) microscopy. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsGΩmsfGFP is a useful tool for membrane imaging and cell-cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and various antibiotic resistance cassettes to facilitate deployment in B. subtilis.IMPORTANCEBacterial cells are fully divided when the new membrane separates the cytoplasm of each daughter. Reproducible staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transporter, PtsG, causes the membrane of Bacillus subtilis to emit bright, uniform fluorescence under a variety of conditions. We use PtsGΩmsfGFP to improve the definition of cytokinesis during microfluidic growth, and we note that a fluorescent PtsG fusion would likely facilitate fluorescent membrane staining in any organism, at least in theory.