Revealing S-layer Functions in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus

The crystalline surface layer (S-layer), consisting of two glycoproteins SlaA and SlaB, is considered to be the exclusive component of the cell envelope outside of the cytoplasmic membrane in Sulfolobus species. Although biochemically and structurally characterized, the S-layer in vivo functions remain largely elusive in Archaea. Here, we investigate how the S-layer genes contribute to the S-layer architecture and affect cellular physiology in a crenarchaeal model, Sulfolobus islandicus M.16.4. Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact, partially, or completely detached SlaA are observed for the ΔslaB mutant. Importantly, we identify a novel S-layer-associated protein M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Additionally, we find that mutants deficient in SlaA form large cell aggregates and the individual cell size varies significantly. The slaB gene deletion also causes noticeable cellular aggregation, but the size of those aggregates is smaller when compared to ΔslaA and ΔslaAB mutants. We further show the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. Finally, we demonstrate that the ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells where the cell cycle is tightly regulated. Together these data suggest that the lack of slaA results in either cell fusion or irregularities in cell division. Our studies provide novel insights into the physiological and cellular functions of the S-layer in Archaea.