Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food-related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterize their surface mechanically. The progression in elasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.Lay Description
Under adverse or challenging environments, certain bacteria are able to transform themselves into a dormant, non-reproductive stage known as spores. These spores once formed, are quite distinct from their “mother cells” with new synthetic protective layers, which causes them to be extremely resilient. Spores are amongst the toughest constructs in nature and can withstand harsh external environmental conditions including high and low temperatures, radiation and chemical disinfectants. These spores have been the subject of several studies owing to their fundamental and applied importance. For instance, several bacterial spores are important as causative agents of anthrax and food borne ailments. Observing the process by which a vegetative cell transforms itself to a spore is key in understanding this important biological phenomena. Here we use a technique called atomic force microscopy (AFM) which essentially probes the exterior surface of the cell using a sharp cantilever. We are therefore able to capture topography and mechanical information at an extremely high spatial resolution (nanometers) and over time. Using the AFM, we can observe single cells and spores under physiological conditions and get precise spatial maps that show how the cell surface is transformed by this process, resulting in the eventual release of a spore. We show interesting changes in the outer surface of the cell during the sporulation process in addition to measurements of the elastic modulus over time. Such techniques have the ability to visualize and understand the early stages of spore development as well as observe such processes in complex samples.