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, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. 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 PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly 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 transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.