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Atomic force microscopy reveals bacterial endospores’ survival tactics

In the winter of 1917, Norwegian police arrested Swedish spy Otto von Rosen. The agent, secretly employed by German intelligence services, was planning to attack farm and other animals of the allied countries with sugar cubes. Inside them, von Rosen had concealed small glass vials with liquid containing Bacillus anthracis. It was the first attempt in history to use this pathogen as a bacteriological weapon, though the plan was thwarted by the arrest of the agent working to favor German interests during World War I. When the police captured von Rosen, they found in his suitcase the sugar cubes containing their dangerous spores. The cubes were kept and guarded for decades in a museum in Trondheim (Norway).

The story of von Rosen might sound like something taken from a spy film. But what is important for this article is that the Bacillus anthracis spores stayed viable for decades. This is proof of the survival capacity of spores, considered the most resistant life forms on Earth. Despite the fascination they arouse, we do not yet know what allows bacterial cells to protect themselves from a lack of nutrients, and produce airtight microcapsules made up of proteins and lipids, that store in their interior bacteria’s genetic matter. The structures enable them to last for prolonged periods in especially adverse environmental conditions. One of the more intriguing unknowns has to do with spore’s capacity to respond to changes in environmental relative humidity, or hygroscopicity, absorbing humidity without compromising their future viability.

The teams of Gabriel Gomila, of the Institute for Bioengineering of Catalonia, and Antonio Júarez, from the University of Barcelona, have analyzed this hygroscopic property of endospores using a technique called electrostatic force microscopy (EFM), a version of atomic force microscopy (AFM). Their main goal was to analyze the internal hydration properties of endospores in conditions of high relative humidity, to better understand the fundamental mechanisms of resistance, and possibly design future technological applications. “We show that the internal hydration properties of single Bacillus cereus endospores in air under different relative humidity (RH) conditions can be determined through the measurement of its electric permittivity,” state the authors of a study published in the ACS Nano journal.

Electrostatic force microscopy images of a single endospore and a of single bacterial cell under low (top) and high (bottom) relative humidity conditions.
Image: electric EFM images of a single endospore and a of single bacterial cell under low (top) and high (bottom) relative humidity conditions. The increased contrast in the images at high relative humidities correlates with the moisture uptake by the endospore and bacterial cell. Source: IBEC.

In the past, scientists had used different approaches to determine endospores’ hydration levels, such as high-resolution secondary ion mass spectrometry (NanoSIMS), confocal Raman microspectroscopy, fluorescence redistribution after photobleaching microscopy (FRAP), automated scanning optical microscopy or microsystem techniques such as single particle levitation and suspended microchannel resonators. Although IBEC and UB researchers underscore the results achieved with these techniques, the lack of spatial resolution, the impossibility to work in situ without destroying the endospore or the low sensitivity to measure the internal hydration properties had limited previous research efforts. As opposed to other methods, electrostatic force microscopy offers the possibility to measure dielectric properties at the local level, an important advantage in the study of samples that were not of biological origin, such as polymer films or nanotubes, and biological-origin samples such as bacterial cells and individual viral particles.

EFM is “sensitive to the internal dielectric properties of the samples, since it is based on the measurement of long-range electric forces, and it is also sensitive to the presence of moisture in the sample, due to the large electric permittivity of water (εr,water∼80),” say the researchers in the article published in ACS Nano. These two characteristics turn electrostatic force microscopy into the ideal technique for in situ testing to analyze endospores without having to destroy the sample. “We have been able to observe how the endospore structure can preserve its core, where the DNA is housed, even when subjected to conditions of low hydration, and regardless of the relative humidity conditions. This key property is added to the rest of endospores’ extraordinary abilities,” states Gomila. This characteristic is fundamental to explain their surprising response to water, which has facilitated development of technological applications with the manufacture of endospore-based energy harvesting devices or electromechanical featuring spores with graphene quantum dots.

The study now being published not only offers details on the “tricks” deployed by endospores to withstand the most adverse environmental conditions, as occurred with the spores sealed inside spy Otto von Rosen’s sugar cubes, it also demonstrates the potential of electrostatic force microscopy to measure the hygroscopic properties of objects at the nanoscale, which could also be useful in the future to study moisture-dependent biological processes. In the mid-term, the EFM technique may help to better understand the production of micotoxins, one of the most significant threats for food safety, or the analysis of the aerosols that nanoparticles carry, and are of interest for atmospheric sciences, to give just two examples.