Structure and function of the archaeal response regulator CheY

Abstract

Motility is a central feature of many microorganisms and provides an efficient strategy to respond to environmental changes. Bacteria and archaea have developed fundamentally different rotary motors enabling their motility, termed flagellum and archaellum, respectively. Bacterial motility along chemical gradients, called chemotaxis, critically relies on the response regulator CheY, which, when phosphorylated, inverses the rotational direction of the flagellum via a switch complex at the base of the motor. The structural difference between archaellum and flagellum and the presence of functional CheY in archaea raises the question of how the CheY protein changed to allow communication with the archaeal motility machinery. Here we show that archaeal CheY shares the overall structure and mechanism of magnesium-dependent phosphorylation with its bacterial counterpart. However, bacterial and archaeal CheY differ in the electrostatic potential of the helix α4. The helix α4 is important in bacteria for interaction with the flagellar switch complex, a structure that is absent in archaea. We demonstrated that phosphorylation-dependent activation, and conserved residues in the archaeal CheY helix α4, are important for interaction with the archaeal-specific adaptor protein CheF. This forms a bridge between the chemotaxis system and the archaeal motility machinery. Conclusively, archaeal CheY proteins conserved the central mechanistic features between bacteria and archaea, but differ in the helix α4 to allow binding to an archaellum-specific interaction partner.

ArlCDE form the archaeal switch complex

“…Corresponding with this hypothesis, an interactome study in Halobacterium salinarum, previously indicated that ArlCE (fused in this organism) interacts with the archaeal specific chemotaxis protein CheF (Figure 1a) (Schlesner et al, 2009;Schlesner et al, 2012). Motile archaea encoding the bacterial-like chemotaxis system, all possess the adaptor protein CheF, which can bind to CheY and as such forms a link between the chemotaxis system and the archaellum (Schlesner et al, 2009;Quax et al, 2018b).…”

An oscillating MinD protein determines the cellular positioning of the motility machinery in archaea

“…Cells were serially diluted and observed at early log-phase. H. volcanii wild type cells are motile and rod-shaped in early log phase and round and non-motile in stationary phase 44,46 . The majority of observed cells of the wild-type performed a random walk in the absence of directional stimuli, as has been observed before 46 (Movie S8).…”

“…Haloferax volcanii was grown and genetically manipulated as described in 46,69 . Depending on the experiment, the cells were grown at 45 °C or 42 °C in either rich YPC medium with Bacto TM yeast extract, peptone (Oxoid, UK) and Bacto TM Casamino acids (BD Biosciences, UK or Oxoid) or in selective CA medium containing only Bacto TM casamino acids 46,69 , occasionally supplemented with a trace elements solution as described 43 was also used where indicated (CAB medium). Plates were incubated at 45°C and liquid cultures were shaken at 120-200 rpm.…”

“…Cell motility was analyzed by live microscopy as described in 46 . Briefly, precultures of cells were grown over night in CA medium with uracil at 45 °C.…”

“…Recent work in H. volcanii has indicated that positioning of the motility structure, chemosensory arrays and cell division machinery is regulated by unknown mechanisms [43][44][45] . H. volcanii cells display different shapes dependent on the growth stage of the culture 43,44,46 . Cells in early log-phase are rod-shaped and motile.…”