Atomic force microscopy (AFM) has shown the alterations of spore coat and germ cell wall that accompany the transformation from a spore to a vegetative cell. Although significant progress has been made in understanding the biochemical and genetic bases of the spore germination process, it is still unclear how a spore breaks out of its dormant state. But a new in vitro study of single-germinating Bacillus atrophaeus spores details how the spore coat structures break down, and it shows with unprecedented resolution how the new bacterium emerges from the disintegrating spore. Emergence of vegetative cells: 60- to 70-nm-deep apertures in the rodlet layer that gradually enlarged (C and D), and subsequently eroded the entire spore coat (E). Germ cells emerged from these apertures.(Photo courtesy Lawrence Livermore National Laboratory) The researchers, including Marco Plomp, lead author at Lawrence Livermore National Laboratory (LLNL), and those from Children's Hospital Oakland Research Institute and Northwestern University, used AFM to identify disassembly of the outer spore coat rodlet structures, which appear to be structurally similar to amyloid fibrils that have been associated with neural degenerative diseases, such as Alzheimer's and prion diseases. Their findings are published in Proceedings of the National Academy of Sciences."A thorough understanding of spore germination is important for the development of new countermeasures that identify the earliest stages of a wide range of spore mediated diseases, including botulism, gas gangrene and pulmonary anthrax," said Alexander Malkin, senior author from LLNL's Biosciences and Biotechnology Div. "But it's also important to gain fundamental insights into the key events in bacterial cell development."Atomic force microscope (AFM) images of the rodlet layer of an intact spore (top), a degrading rodlet layer during germination (middle) and the cell wall structure of the newly emerging vegetative cell (bottom). (Photo courtesy Lawrence Livermore National Laboratory) "The extreme physical and chemical resistance of Bacillus spores suggests that evolutionary forces have captured the mechanical rigidity and resistance of these amyloid self-assembling biomaterials to structure the protective outer spore surface," Plomp said. When exposed to a solution that triggers germination, nanometer-sized etch pits were seen developing in the rodlet layer. These etch pits evolved into ever widening fissures, leaving narrow strips of remaining rodlet structure. In the end, 1- to 3- nm-wide fibrils remained. The in vitro AFM imaging also revealed the porous fibrous cell wall structure of newly emerging and mature vegetative cells, consisting of a network of nanometer-wide peptidoglycan fibers. "These results show that dynamic AFM is a promising tool to investigate the formation and evolution of the bacterial cell wall," Malkin said. The research is funded by LLNL's Laboratory Directed Research and Development program, the Defense Advanced Research Project Agency (DARPA) and the Federal Bureau of Investigation. For more information, visit: www.llnl.gov