These are the largest endospores described thus far, with the largest being over times larger than a Bacillus subtilis endospore. The formation of endospores may help maintain the symbiotic association between these Epulopiscium -like symbionts and their surgeonfish hosts. Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night.
The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. The fish may also benefit from this relationship because it is able to maintain stable microbial populations that assist in digestion and may receive a nutritional gain from microbial products released during mother cell death and spore germination. Endospore formation in some Epulopiscium -like symbionts follows a daily cycle: A Polar septa are formed at the poles of the cell.
B Forespores become engulfed. C Forespores gradually increase in size within the mother cell through the day. D In late afternoon, final preparations for endospore dormancy. E Endospores mature and remain dormant throughout most of the night. F Just before sunrise, the endospores germinate and are released from mother cell to repeat the cycle.
Google Tag Manager. Bacterial Endospores. Endospore Structure The resilience of an endospore can be explained in part by its unique cellular structure. Endospore Development The process of forming an endospore is complex. The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA, and the developing structure is now known as a forespore.
Calcium dipicolinate is incorporated into the forespore during this time. Next the peptidoglycan cortex forms between the two layers and the bacterium adds a spore coat to the outside of the forespore.
Sporulation is now complete, and the mature endospore will be released when the surrounding vegetative cell is degraded. While resistant to extreme heat and radiation, endospores can be destroyed by burning or by autoclaving.
An indirect way to destroy them is to place them in an environment that reactivates them to their vegetative state. They will germinate within a day or two with the right environmental conditions, and then the vegetative cells can be straightforwardly destroyed.
This indirect method is called Tyndallization. It was the usual method for a while in the late 19 th century before the advent of inexpensive autoclaves. Prolonged exposure to ionising radiation, such as x-rays and gamma rays, will also kill most endospores. Reactivation of the endospore occurs when conditions are more favourable and involves activation, germination, and outgrowth.
Even if an endospore is located in plentiful nutrients, it may fail to germinate unless activation has taken place. This may be triggered by heating the endospore. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation.
It is commonly characterised by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress.
As a simplified model for cellular differentiation, the molecular details of endospore formation have been extensively studied, specifically in the model organism Bacillus subtilis. These studies have contributed much to our understanding of the regulation of gene expression, transcription factors, and the sigma factor subunits of RNA polymerase. This is not unexpected, since much of the spore germination machinery and architecture providing spore resistance are universal in all Firmicute spore-formers 2 , 5.
Indeed, pan-species universality was recently shown for the crystalline organization of the DNA in the dormant spore which is an important factor for providing DNA resistance 9. However, while sCMMs were found in spores of all species, their section profiles were somewhat different in different species. In spore of B. Ultimately, analysis of the sCMM 3D structure at higher resolution is needed to get more information about these membranes and their potential link to the CM.
Cryo-tomography of slices prepared by cryo-focussed ion beam milling 23 , 24 , 25 may help to solve this issue when this technique becomes more widely available. So far, we can only speculate about the 3D structure of the sCMMs. The most intuitive fit with the sectioning profiles detected by CEMOVIS this technique gives the most reliable structural representation suggests a 3D structure of a compressed vesicle or tubule Fig.
This hypothesis would be in accordance with known membrane dynamics in cells mediated by fission and fusion of vesicles or small tubules. A potential compression of sCMM bound structures can be explained by the exceptional dryness of the core in association with the deposition of CaDPA which together may deform small membrane-bound compartments like vesicles or tubules.
Future investigation should also focus on the formation of sCMMs during sporulation to understand their positioning and shape within the dormant spore. Sporulation was monitored by inspection with phase-contrast microscopy. The resulting pellet showed three layers from which the uppermost layer was harvested and suspended with 0. The purity of the suspension was checked by phase-contrast microscopy and levels of spore particles were determined using a hemocytometer according to Neubauer.
For sporulation, the agar plates were transferred to room temperature. The progress of sporulation was monitored by phase-contrast microscopy and took a few days to reach significant values.
Cells were harvested using a Drigalski spatula and were filtered through glass wool before they were washed three times with deionized water to remove vegetative cells or debris. Spores of B. Spore pellets of B. The resulting line plot grey level relative to the position on the selection line was used for measuring the distance between minima darkest parts of the selected profile which correspond to the outer electron dense parts of the CM Supplementary Fig.
Line profiles across double-ring membrane-like structures revealed three minima, and the distance between the outer two minima was determined Supplementary Fig. The procedures for conventional thin section electron microscopy were described in detail previously 9. After embedding in an agarose gel, samples were quickly dehydrated and embedded in LR White which then was polymerized on ice by the help of an accelerator.
Sections were stained with a solution of 0. To facilitate image alignment, sections were coated with gold particles at the back side of the supporting film. After immunolabelling, sections were dried before they were contrasted using a solution of 0.
Conventional transmission electron microscopy was done as described above. Quantification of the immunogold labelling was done to assess specificity of the labelling. In this analysis, labelling by the anti-SpoVAD antiserum was compared with the labelling by the three control antibodies anti-rPBP, anti-GFP, anti-Acz2 , which are not known to detect any bacterial protein. For direct comparison, antibody concentrations of all antibodies were adjusted to give similar background levels over the coat layers, which most likely do not contain the target antigen SpoVAD Labelling densities gold particles per membrane length or core area were determined for three different core zones Supplementary Fig.
The detailed procedures of sampling and determination of labelling densities are described in the Supplementary Methods section. The concept of this analysis is to determine the unspecific labelling of structures by control antibodies, which are not know to have any specific affinity for proteins in spores, and to use this labelling as a reference for the labelling with the specific anti-SpoVAD antibody. Any specific labelling must be different from the labelling achieved by the three control antibodies.
The larger the difference is, the more likely the specificity of the labelling is. The variability of the labelling generated by the three different control antibodies, expressed as standard deviation SD of the mean labelling densities determined for each control antibody, provides a measure to assess the difference between control antibody labelling and anti-SpoVAD labelling.
Usually a difference between two distributions is considered significant if the mean values differ at least by 2-fold SD which means that the two distributions would still overlap. A difference of about 5-fold SD exclude a significant overlap of distributions and is therefore considered as a robust criterion to conclude that there is a significant difference between two observed experimental conditions. In particle physics, the 5-fold SD threshold is widely used as a standard because of many false decisions using lower thresholds and it is discussed to use this strict threshold also in biomedicine We therefore computed the difference between labelling densities of anti-SpoVAD and control antibodies in units of the observed SD for the control antibody labelling and assessed the results according to the considerations stated above.
At defined time points see result section spore suspensions 0. To determine the relative core size of dormant and germinating spores, section profiles of spores were randomly selected and photographed 20 cross sections and 10 longitudinal sections for each sample.
Relative core size was calculated by dividing core area by the area covered by the entire spore section. McKenney, P. The Bacillus subtilis endospore: assembly and functions of the multilayered coat.
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