The structure of the periplasmic FlaG-FlaF complex and its essential role for archaellar swimming motility.

Motility structures are vital in all three domains of life. In Archaea, motility is mediated by the archaellum, a rotating type IV pilus-like structure that is a unique nanomachine for swimming motility in nature. Whereas periplasmic FlaF binds the surface layer (S-layer), the structure, assembly and roles of other periplasmic components remain enigmatic, limiting our knowledge of the archaellum's functional interactions. Here, we find that the periplasmic protein FlaG and the association with its paralogue FlaF are essential for archaellation and motility. Therefore, we determine the crystal structure of Sulfolobus acidocaldarius soluble FlaG (sFlaG), which reveals a ß-sandwich fold resembling the S-layer-interacting FlaF soluble domain (sFlaF). Furthermore, we solve the sFlaG2-sFlaF2 co-crystal structure, define its heterotetrameric complex in solution by small-angle X-ray scattering and find that mutations that disrupt the complex abolish motility. Interestingly, the sFlaF and sFlaG of Pyrococcus furiosus form a globular complex, whereas sFlaG alone forms a filament, indicating that FlaF can regulate FlaG filament assembly. Strikingly, Sulfolobus cells that lack the S-layer component bound by FlaF assemble archaella but cannot swim. These collective results support a model where a FlaG filament capped by a FlaG-FlaF complex anchors the archaellum to the S-layer to allow motility.

Cell Structure Changes in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus Lacking the S-Layer.

Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the ΔslaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which 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 show the key physiological and cellular functions of the S-layer in this archaeal cell.IMPORTANCE The S-layer is considered to be the sole component of the cell wall in Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

The essential genome of the crenarchaeal model Sulfolobus islandicus.

Sulfolobus islandicus is a model microorganism in the TACK superphylum of the Archaea, a key lineage in the evolutionary history of cells. Here we report a genome-wide identification of the repertoire of genes essential to S. islandicus growth in culture. We confirm previous targeted gene knockouts, uncover the non-essentiality of functions assumed to be essential to the Sulfolobus cell, including the proteinaceous S-layer, and highlight essential genes whose functions are yet to be determined. Phyletic distributions illustrate the potential transitions that may have occurred during the evolution of this archaeal microorganism, and highlight sets of genes that may have been associated with each transition. We use this comparative context as a lens to focus future research on archaea-specific uncharacterized essential genes that may provide valuable insights into the evolutionary history of cells.