A new fluorescence-based approach for direct visualization of coat formation during sporulation inBacillus cereus

Abstract

AbstractThe pathogenic bacteriaBacillus cereus,Bacillus anthracisandBacillus thuringiensisform spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in liveB. cereuscells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes duringB. cereussporulation. We used these markers to characterize a subpopulation of engulfment-defectiveB. cereuscells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organismBacillus subtilis, the activity of σKis not strictly linked to the state of forespore development inB. cereus.