Model System Mimicking the Cell Envelope of Archaea

Archaea are one of the oldest life-forms existing on Earth. These unicellular organisms are often adapted to extreme habitats. Many representatives of archaea are capable to live at very high temperatures (i.e., above 80 C), very low or high pH values, high salt concentrations or high pressures. Since the cell envelope of many archaea consists only of a very thin layer of fat (lipid membrane), into which an outermost crystalline protein layer is anchored, the question arises how Nature can accomplish this high resistance to extreme environmental conditions. The project "Generation and characterization of artificial archaeal cell envelope structures and their relevance as model membrane platforms" will study the reassembly of cell envelopes of archaea using previously isolated biological components, i.e., lipids and proteins. It aims to clarify the question how the self-organization of etherlipids and surface layer proteins proceeds in detail and which anchoring strategies are available for the formation of an artificial cell membrane. In addition, the question is addressed which properties of the biomolecules themselves and their assembly into macroscopic cell envelopes cause this amazing resistance to the extreme habitat conditions. Hence, selected archaea strains will be bred in the bioreactor and subsequently the basic building blocks, which are surface layer proteins and fats (so-called etherlipids) will be isolated from the biomass. In addition, the surface layer proteins can also be genetically produced by host cells. Moreover, if desired anchoring groups for the specific binding to etherlipids can be genetically inserted. This is a new approach, which has previously not attempted by another research group. By the application of Natures construction principle, the cell envelope structure of archaea will be reconstructed layer by layer. Each step will be tracked and analyzed using modern microscopial and surfaces sensitive techniques. Next to high- resolution light and fluorescence microscopy also electron and atomic force microscopy will be used as imaging methods. The main surface-sensitive methods include surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. These methods are used for determining the morphology, thickness in the nanometer range and quality of each layer. The results of this project will provide valuable insights into the isolation and in particular the self- assembly of cell envelope components. This knowledge can be applied to produce surface coatings with very specific properties (e.g., anti-fouling or self-cleaning). Further fields of application are biomimetic membrane systems, which can be used to study and model the cell walls of archaea. The latter have also a great potential to serve as model systems into which membrane-active peptides and membrane proteins can be incorporated and systematically investigated. In future it might be even possible to develop highly sensitive diagnostic and sensor systems.

Within the project "Biomimetic model system of the cell wall of archaea" different archaea were cultivated to extract two essential membrane components from the biomass. On the one hand, ether lipids were extracted from the plasma membrane of the organisms, and on the other hand, attempts were made to isolate surface proteins, the so-called S-layer proteins. Both building blocks are very interesting because, as outermost contact surfaces of the archaea, they can withstand extreme living conditions such as temperatures up to 120 C and a very acidic pH and must therefore be very stable building blocks. It was possible to reproducibly obtain a desired lipid composition of the plasma membrane by systematically adjusting different cultivation conditions. Planar and spherical membranes were prepared from the extracted ether lipids, characterized, and used as binding matrices for bacterial S-layer proteins. The archaeal S-layer proteins could not be detached from the archaea as individual proteins by either chemical, physical, or genetic methods. However, using a genetic approach it was shown that the S-layer is very important for cell division and maintenance of cell shape. It was also interesting that ultrafiltration membranes comprising of isolated S-layer protein fragments attached to microfilters could be made. These may have applications in biotechnology as filters, but also as a simplified model to understand the biochemistry and biophysics of S-layer proteins. A literature review was conducted as part of the project to provide an overview of the current state of Archaea biotechnology. This included a description of the current state of research and development and industrial use of archaeal cell factories, their role and potential for the future of sustainable bioprocessing, and their physiological and biotechnological potential. To objectively analyse the progress of the technologies in question, the established Technology Readiness Level (TRL) scale was adapted for specific application in microbial biotechnology. The new Bio-Technology Readiness Level (B-TRL) scale was defined for this review and applied throughout. This now makes it for the first time possible to objectively compare the B-TRL for different archaeal cell factory products. The project was of relevance not only for (bio)nanotechnology and biophysics but advanced also related areas of research like ecophysiology, geo-, astro-, and biotechnology. The knowledge on how to choose proper growing conditions to obtain a desired lipid profile and scale-up parameters is beneficial for research fields like cosmetics and drug delivery. Finally, the project findings opened a promising new approach for membrane protein-based biosensors in areas ranging from medicine over diagnostics to environmental monitoring.