Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped (‘plate’) cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace element micronutrients. With these supplemented media, transient development of plate cells into uniformly shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies (‘pleomorphic rods’) were observed at all growth stages of the culture; the highly elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early log-phase rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results reveal the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

18Some microbes display pleomorphism, showing variable cell shapes in a single 19 culture, whereas others differentiate to adapt to changed environmental conditions. 20The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped 21 ('plate') cells in culture, but may also be present as rods, and can develop into motile 22 of archaeal cell biology. 50Archaea exhibit some of the most diverse and unusual microbial cell morphologies. 51Cell shapes range from rods and cocci to striking triangles and squares. However, 52little is known about the mechanisms and environmental cues that dictate archaeal cell 53 morphology, or the regulation of transitions between cell morphologies. The model 54 haloarchaeon, Haloferax volcanii, when first isolated, was described as mainly disk-55shaped cells, with cell shape and size varying significantly (1). In routine liquid 56 cultures of H. volcanii, both pleomorphic discoid (plate) and rod cell morphologies 57 may be observed, occasionally with more unusual shapes (2-4). 58The conditions that influence H. volcanii cell shapes and the relative abundance of the 59 distinct types are not well understood, and specific signals have not been identified. In 60 H. volcanii biofilms, substantial elongation (filamentation) has been observed in a 61 subpopulation of cells (5). Furthermore, H. volcanii forms rods in swimming-motility 62 soft-agar, such that rods are observed at the forefront of expanding colonies of 63 swimming cells (6). The tubulin-like cytoskeletal protein CetZ1 was required for rod-64formation, suggesting a connection between morphology and motility that may be 65 expected based on improved hydrodynamics or directional movement of rods (4, 6). 66Conversely, the peptidase archaeosortase A (ArtA) and phosphatidylethanolamine 67 biosynthesis enzymes PssA and PssD, which are required for the C-terminal 68 processing and covalent lipid attachment of several H. volcanii surface proteins 69 including the surface (S-layer) glycoprotein, are also required for effective and stable 70 plate-shaped cell formation (7, 8). 71To better understand the regulation of cell shape switching and the importance of the 72 distinct shapes, we aimed to define conditions that can be robustly used to study these 73 processes. We determined that inclusion of a trace elements (TE) solution in both 74 complex and semi-defined culture media substantially improves growth and culture 75 reproducibility for this species. In the new media, early-log rod formation was also 76 more reproducible, while the pleomorphic rods and other shapes seen in cultures 77 lacking TE were absent. We further defined culture conditions and characterized these 78 CetZ1-dependent morphological changes, which may serve as experimental models 79 for studies of morphological development in archaea. 80

The tubulin superfamily of cytoskeletal proteins is widespread in all three domains of life — Archaea, Bacteria and Eukarya. Tubulins build the microtubules of the eukaryotic cytoskeleton, whereas members of the homologous FtsZ family construct the division ring in prokaryotes and some eukaryotic organelles. Their functions are relatively poorly understood in archaea, yet these microbes contain a remarkable diversity of tubulin superfamily proteins, including FtsZ for division, a newly described major family called CetZ that is involved in archaeal cell shape control, and several other divergent families of unclear function that are implicated in a variety of cell envelope-remodelling contexts. Archaeal model organisms, particularly halophilic archaea such as Haloferax volcanii, have sufficiently developed genetic tools and we show why their large, flattened cells that are capable of controlled differentiation are also well suited to cell biological investigations by live-cell high-resolution light and electron microscopy. As most archaea only have a glycoprotein lattice S-layer, rather than a peptidoglycan cell wall like bacteria, the activity of the tubulin-like cytoskeletal proteins at the cell envelope is expected to vary significantly, and may involve direct membrane remodelling or directed synthesis or insertion of the S-layer protein subunits. Further studies of archaeal cell biology will provide fresh insight into the evolution of cells and the principles in common to their fundamental activities across the full spectrum of cellular life.