Archaeal tubulin-like proteins CetZ1 and CetZ2 have opposing effects on cell morphology during the growth cycle ofHaloferax volcanii

CetZs are archaeal tubulin superfamily cytoskeletal proteins implicated in the control of cell shape and motility. In the pleomorphic archaeon Haloferax volcanii , CetZ1 is required for the transformation of a discoid, or plate-like, cell morphology to the rod shape during early-mid log phase cultures and for the development of swimming motility. In this study, we found that the paralog CetZ2 is not required for rod development or reversion to plates in log phase but was strongly upregulated later in stationary phase, where it promotes the maintenance of plate cell shape. CetZ2 cytoskeletal structures visualized through the production of a functional fluorescent tagged CetZ2 were most dynamic specifically in mid-stationary phase, where they showed directional movement around the cell edge and other complex cytomotive-like behaviours. Furthermore, in mid-stationary phase, the dynamics of CetZ1 and CetZ2 cytoskeletal structures were specifically dependent on the presence of one another and their GTPase activities that control the polymerization-depolymerization cycle. Together, the results suggest that CetZ2 counteracts the CetZ1-based rod development pathway to maintain plate shape, and they imply that additional stationary-phase factors are also involved. CetZ1 and CetZ2 are co-conserved in many haloarchaea, suggesting that their coordinated functions at different stages of the growth cycle are widely utilized to control cell shapeshifting. More generally, our findings show that CetZ paralogues can have distinct and non-redundant functions, indicating that diversification and specialisation within the tubulin superfamily has occurred multiple times in archaeal evolution. Significance This study describes the first defined function of a CetZ2 protein, which represents a distinct haloarchaeal protein family within the near-universal tubulin superfamily of cytoskeletal proteins. Using the model archaeal organism Haloferax volcanii , we found that CetZ2 maintains the plate or disk-like cell morphology specifically in the stationary phase of the growth cycle, by counteracting CetZ1-based rod development. We also showed for the first time that CetZ2 can form dynamic cytoskeletal filaments that show directional movement around the cell in mid-stationary phase. The CetZ1-CetZ2 interplay we detected specifically at this stage may represent a paradigm for understanding the evolution of antagonistic cytoskeletal functions, such as those that led to a reliance on active and inactive subunits during the early evolution of microtubules in eukaryotes.