by Alise Muok and Ariane Briegel
Try to imagine a creature that can survive from the heat of 150°C (300°F) to the cold of –270°C (–454 °F), in the absence of oxygen, after complete dehydration, at 1,000× atmospheric pressure, and after being exposed to the radiation and vacuum of space. Who might such space travelers be? Given their widespread news coverage, including a recent post by Elio, your first thought might be tardigrades. But there is a different type of creature that can also survive the extreme conditions of space.
That's right, we are talking about bacterial spores! Like tardigrades, many bacteria respond to stress conditions by going into a dormant yet resilient state that can revert back into an active state when their surroundings become more favorable. While tardigrades undergo these transitions by regulating specific metabolic processes, bacteria produce spores that can germinate. These spores contain the identical genetic information of their parent cell but they are coated in a thick layer of peptidoglycan (PG) and proteins that protect them from the environment. When environmental conditions are met, the spore PG coat will disassemble to produce an active bacterial cell once again. Because of their protective coat, the spores can survive in environments the parent cell could not, including the harsh conditions of space.
The rearrangement of PG layers during sporulation is a complex process that has been studied extensively in the bacterial model organism Bacillus subtilis by microscopy techniques, which allow researchers to directly view and image PG layers in vivo. Using these data, researchers have seen that sporulation occurs in four stages. In the first and second stages, the mother cell replicates its genome and then asymmetric septation of the cell occurs to separate the genomes into two different compartments – the smaller of these will eventually become the spore. In the third stage the septum starts to curve around the forming spore (called the forespore) until it is completely engulfed inside the mother cell (see here in STC). In the final stage, the forespore becomes surrounded by thick layers of PG and then is released from the mother cell. However, several aspects of this system have remained poorly understood due to limitations of microscopy techniques. In an exciting leap forward to understanding this process, new microscopy data presented in Khanna et al. (Open Access PDF) elucidate important features of PG synthesis and degradation during sporulation in B. subtilis. This advance was made possible by the use of a technique called cryo-electron microscopy (cryo-EM) that allowed these researchers to image sporulating cells at the highest resolution reported thus far.
Before the invention of electron microscopes, scientists were able to see living things with light microscopes, but could not see inside cells at a molecular resolution due to the limiting wavelength of visible light. However, the wavelength of an electron is about 100,000× shorter than that of visible light, revealing structural details at an unprecedented level. To visualize the power of an electron microscope in your mind's eye, imagine that we got so frustrated with writing this article that we asked our friends at NASA to shoot it to the surface of the moon (next to the dormant water bear colony located in the 'sea of serenity', 32.595°N, 19.349°E). An electron microscope has enough magnifying power that we would still be able to read the article from here.
While electrons have a short wavelength that makes them ideal for high-resolution imaging, the electrons can only be used for imaging if the sample is contained in a vacuum, thus preventing the use of any sample that contains water – which basically means anything biological. In the past, this issue was circumvented by the development of elaborate sample preparation workflows that typically included sample dehydration, heavy metal staining, plastic embedding, and sectioning. This allowed imaging of a wide variety of biological specimens and allowed researchers to gain great insight into cellular biology. However, the invasive nature of the preparation procedures affects the delicate ultrastructure of cells and prevents more detailed insight into structures at the macromolecular level. But then cryo-EM came to the rescue! Instead of invasive preparation procedures, biological samples are simply plunged into a liquid cryogen. Freezing of the sample happens so fast that water molecules are unable to form crystals, and the specimens are embedded in a glass like, vitreous ice. Unlike crystalline ice, vitreous ice does not damage the sample in any way so the biological samples will retain native macromolecular structures. Furthermore, to study microbes in three dimensions by cryo-EM, we can simply tilt our specimens in the microscope while taking multiple images with different orientations of the sample relative to the electron beam and then computationally back-project these images to generate a 3D image. This is similar to doing a CAT scan, but in this case we rotate the sample and not the microscope. This method is called cryo-electron tomography (cryo-ET) and has emerged as a powerful tool to study the structure and function of macromolecular complexes inside of cells.
Before the Khanna et al. paper, research published by Tocheva et al. in 2013 also utilized cryo-ET to provide great insight into the process of sporulation in B. subtilis. They not only saw all the stages of sporulation in this organism but also deduced that a thin PG layer remains around the forespore during engulfment. It had been previously thought that the PG layer would completely disassemble to accommodate the bending force needed for engulfment. However, the resolution achieved in that study was not sufficient to unravel finer structural details. This limitation resulted from the fact that spores and whole bacterial cells are very large objects for cryo-ET, which is rather limited by the thickness of samples that can be imaged. Just like with light microscopy, if your sample is very thick, objects will just appear as dark silhouettes without any internal detail. Instead, the electrons must be able to penetrate through the sample to provide an image. In practice, samples of around half of a micrometer or less are suitable, while a thicker specimen is challenging or even impossible.
Spores are one to two micrometers in diameter, and surrounded in a dense PG coat… So how was the team of Khanna et al. able to uncover the finer details of spore formation in B. subtilis using cryo-ET? They were able to achieve this by using another new instrument in the cryo-EM toolbox called 'cryo-focused ion beam milling' (cryo-FIB). Essentially, this method lets you use an ion beam that is so precise that you can cut cells into layers that are thin enough for cryo-ET experiments. After vitrifying thicker samples such as whole B. subtilis cells undergoing sporulation, the sample is loaded in the cryo-FIB instrument. Here, a gallium ion beam is used to cut thin 'windows' into the otherwise too thick sample. The sample is then transferred into a cryo-EM scope, where tomograms (using cryo-ET) of high quality can be collected at the positions of these 'windows'. So to put things more precisely, Khanna et al. didn't just use an electron microscope to elucidate sporulation at unparalleled detail, they first used cryo-FIB to make their samples thinner and then used cryo-ET to generate 3D models of these thin slices (Figures 2+3).
Using this workflow, Khanna et al. revealed the organization of PG around the forespore in greater detail. Specifically, they were able to see that the PG layer is continuously synthesized and degraded at the leading edge of the mother cell during engulfment. This allows the septum to be 'pushed' around the forespore and supply the force needed for engulfment. Furthermore, the resolution of these data was so high that they were able to resolve tiny finger-like projections of the mother cell membrane at the leading edge that forms around the forespore. Their formation depends on an enzyme complex called DMP. It is formed by the proteins SpoIID, SpoIIM and SpoIIP, which together have PG degrading activity. This complex is tethering the membrane to the PG and degrading it ahead of the engulfing membrane. The authors further suggest a functional role of the fingerlike protrusions, which may provide the membrane a tighter lateral grip during its movement by serving as a ratchet that prevents backward sliding.
Since the relatively recent introduction of cryo-EM, emerging methods have increasingly lead to astounding discoveries of complex biological processes. We can now see inside organisms that are literally frozen in time in details we previously thought impossible. And for all this we need to thank the high-tech process that is cryo-EM imaging. Imagine, your samples are not just put under a lens to magnify your vision, but they are put inside a vacuum sealed column, at temperatures below –170°C, and then exposed to high amount of radiation from an electron source to generate an image that exposes the function of molecules. Thank you, cryo-EM! And since these conditions are very similar to those in space, it makes us wonder if water bears and bacterial spores could be 'revived' after imaging them in a cryo-EM scope. So the next time you see fan art of a water bear in space, imagine a bacterial spore friend in accompaniment!
Ariane is a professor at Leiden University in the Netherlands. She has nearly 20 years of experience using cryo-electron microscopy to study the ultrastructure of microbes. Her lab focuses on investigating how microbes sense and respond to their environment and explores the structures and functions of the molecular complexes involved in these behaviors. Alise is a postdoc in Ariane Briegel's lab at Leiden University. She has a background in structural biology, specifically in analysing protein systems in bacteria. When they are not in lab, Alise loves to paint and practice aerial silks. Ariane loves gardening, hiking or learning how to do aerial silks from Alise!