Many Archaea love extreme habitats. They can be found in hypersaline lakes, environments of extremely high or low pH, underneath the ice of the arctic or in volcanic hot springs. Our lab pet, Sulfolobus solfataricus is an extremely thermophilic crenarchaeon, isolated in 1980 from the bubbling center of an 80°C-hot, acidic and sulfuric spring in Pozzuoli/Italy1.
Sampling from the hot spring in Pozzuoli, Italy, from which Sulfolobus solfataricus was originally isolated. Kevin Pfeifer (left) and Christa Schleper (right) in September 2018. © Schleper Lab
To be able to thrive in such harsh environmental conditions, Sulfolobus must literally have a “thick skin”. This is indeed the case, as in an electron microscope, a symmetrically arranged cell wall surrounding the individual cell like a protective chainmail is visible. The so-called S-layer (“surface layer”) consists of at least two highly glycosylated protein species which assemble into regular arrays, forming the sole rigid envelope covering the Sulfolobus cell2. Since it is widespread among the archaea and found in some bacteria, the S-layer might represent an early invention of life and probably decorated the first microorganisms on Earth. This suggests that it plays an important role, potentially even beyond the function of the protective shell.
Sulfolobus solfataricus cell covered by S-layer (scanning electron micrograph: SEM, transmission electron micrograph: TEM). The S-layer consists of two highly glycosylated proteins: SlaA (yellow) and a SlaB (orange), the latter forming the stalk anchored in the cell membrane. © Kevin Pfeifer.
Hooked by its enigmatic function, the Christa Schleper Lab (University of Vienna) and Bernhard Schuster Lab (University of Natural Resources and Life Sciences, Vienna) joined forces to decipher the physiological role of the S-layer in Sulfolobus by analyzing phenotypes of S-layer stripped cells in vivo. After several unlucky attempts to chemically remove the S-layer, we decided to make use of our hot-out-of-the-oven gene silencing tool to gradually “CRISPR the S-layer away”. The silencing technology is based on an endogenous CRISPR type III endonuclease which can be hijacked by heterologously expressed synthetic small RNAs that guide the complex to matching sites on a desired host mRNA, which is subsequently degraded. As in past experiments, the tool had only been used to silence non-essential reporter genes in S. solfataricus3, Isabelle was eager to finally apply the tool on an interesting host gene – slaB encoding the S-layer anchor.
The CRISPR approach worked instantly and slaB was silenced to up to 75%. After some days of incubation at 80°C and shaking conditions, mimicking the Sulfolobus environment, the first distinct phenotype revealed itself: S-layer silenced cultures suffered from strong growth retardation. The excitement grew when we looked at them more closely: “These cells are freaking huge and the S-layer is peeling - off like a banana peel”, were Kevin`s first words when he came back from the electron microscope. Our excitement finally peaked when we found that these partially stripped cells were not only up to fivefold bigger than the wild type, but also often carried an increased number of genome copies. The case was clear: Sulfolobus solfataricus could not divide properly without an intact S-layer.
Overview of a Sulfolobus culture (scanning electron micrographs) of control (left) and S-layer depleted (right) cells shows striking size differences in slaB silenced cultures (upper panel). The cross section (transmission electron micrographs of thin sections) of a Sulfolobus cell shows a clear S-layer (left), which is destabilized in S-layer depleted cells and peels off the cell (lower panel). Adapted from Zink et al., 2019, NatComm.
We further set out to investigate if S-layer depletion had an effect on virus infection. We were astonished, as contrary to our expectations, the S-layer depleted cells were less efficiently infected by virus SSV1 than the wild type cells. After endless rounds of infection assays using different virion amounts, we had to start believing: Instead of constituting a virus barrier, the S-layer seemed important for the efficient infection of cells by SSV1.
Our S-layer journey started in 2016 and lasted until 2019. Conclusively, it was a very exciting and coffee-intense project with great revelations and interesting twists and turns. Most excitingly, S-layer mutant cells of the closely related strain S. islandicus were recently reported to grow quite happily - without strong growth defects - in cell clusters4. This indicates that the dependency on the S-layer may strongly vary with the environmental conditions and among different strains, as with a maximum of 75% silencing, it seemed to be indispensable for the growth of S. solfataricus with planktonic life style. We have the feeling that more revelations are going to be made about archaeal cell coats in the future.
1. Zillig, W. et al. The Sulfolobus-"Caldariella" group: Taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch. Microbiol. 125, 259–269 (1980).
2. Veith, A. et al. Acidianus, Sulfolobus and Metallosphaera surface layers: Structure, composition and gene expression. Mol. Microbiol. 73, 58–72 (2009).
3. Zebec, Z., Zink, I. A., Kerou, M. & Schleper, C. Efficient CRISPR-Mediated Post-transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression. G3: Genes|Genomes|Genetics 6, 3161–3168 (2016).
4. Zhang, C. et al. Cell Structure Changes in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus Lacking the S-Layer. MBio 10, e01589-19 (2019).