In vitro high-resolution structural dynamics of single germinating bacterial spores

Abstract

Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here, we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by NMR. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.atomic force microscopy ͉ cell wall ͉ germination ͉ amyloid ͉ peptidoglycan W hen starved for nutrients, Bacillus and Clostridium cells initiate a series of genetic, biochemical, and structural events that result in the formation of a metabolically dormant endospore (1). Spores can remain dormant for extended time periods and possess a remarkable resistance to environmental insults (i.e., heat, radiation, toxic chemicals, and pH extremes) (1-5) that are lethal to vegetative cells. The resistance and persistence of dormant spores is attributed to a multilayer spore architecture (6). Upon exposure to favorable conditions, spores break dormancy through the process of germination (2,7,8) and eventually reenter the vegetative mode of replication.A comprehensive understanding of the mechanisms controlling spore germination is of fundamental importance both for practical applications related to the prevention of a wide range of diseases by spore-forming bacteria (including food poisoning and pulmonary anthrax), as well as for fundamental studies of cell development. Germination involves an ordered sequence of chemical, degradative, biosynthetic, and genetic events (2, 8).Significant progress has been made in understanding the biochemical and genetic bases for the germination process (2). Germination is triggered by the interaction of germinants with specific receptors (2, 7, 9) in the inner spore core membrane, causing the release of the dipicolinic acid and its replacement by water. Subsequent hydrolysis of the spore cortex, further uptake of water, core expansion, and spore coat hydrolysis allow emergence of the incipient vegetative cell.The role of the spore coat in the germination process is unclear (2, 6) and is the focus of this study. Spore coat structure regulates the permeation of germinant molecules (7-10). It is believed that penetration of germinants proceeds through pores in the coat structure and may involve GerP proteins (9).Atomic force micro...

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

“…Surface structures on various types of cells were visualized using conventional AFM. High-resolution images were achieved mainly on microbial cells, [8][9][10] while high-resolution images of animal cells remain challenging. 36 This difference may be due to the softness of animal cells.…”

“…6,7 In particular, visualization of the architecture of a living cell is a novel powerful approach to unveil the molecular organization in vivo. For example, the hexagonal S-layer of Corynebacterium glutamicum, 8 the peptidoglycan organization of Lactococcus lactis, 9 and the germinating spores of Bacillus atrophaeus 10 have been directly visualized in living microbial cells using AFM.…”

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

“…Significant advances have been made in recent years through the application of mainly genetic techniques to characterize the molecular apparatus involved in spore germination, identifying structural genes that encode germinant receptors (4,8,14,23,37), ion channels (31,35), and hydrolytic enzymes involved in the degradation of the cortical peptidoglycan that surrounds the spore protoplast (6,11,16,21). Cytological observations on spore coat degradation during germination (29) have also recently been extended by the application of atomic force microscopy to provide insights to the structural basis for the disassembly of the outer layers that provide the primary barrier to environmental insult (26).…”

Coherent X-ray diffractive imaging on the single-cell-level of microbial samples

“…For instance, comparing quantitative three-dimensional density maps of the endospores in a dry and fully hydrated state may further elucidate their water content which is important for their heat resistance [283]. Endospores may also be candidates for observing time snapshots of dynamical processes such as hydration or germination [230,232] which are di cult to observe otherwise. F{f g}( ν) =f ( ν) ·g( ν), (1.141) or F{f · g}( ν) =f g ( ν).…”

“…The full process ending in a free endospore takes about 7-8 hours in case of B. subtilis [180,229]. Vegetative growth is continued after germination of the endospore [205,230,272].…”