Improved growth and morphological plasticity ofHaloferax volcanii

Abstract

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

Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeonHaloferax volcanii

“…Cells growing in Hv-Cab medium (Figure 3a, left) have irregular or round shapes and most cells possess single rings at mid cell. In contrast, cells cultivated in YPC medium (Figure 3a, right) are more often rod-shaped (Delmas et al, 2013;Duggin et al, 2015;de Silva et al, 2020), their FtsZ1 structures appeared more diverse and FtsZ1 rings are slightly thicker. Next, we quantified these observations (Figure 3b and 3c).…”

“…We created the stable deletionmutant strain WR806 and compared it with the red-colored H. volcanii laboratory strain H119 which synthesizes carotenoids (Figure 1b). Cultured in colorless Hv-Cab medium (Duggin et al, 2015;de Silva et al, 2020), WR806 appeared white on agarose plates and hardly distinguishable from background noise under SMLM imaging conditions, while H119 colonies were red-colored on plates and the cells exhibited an autofluorescence signal. This and the fact that the deletion did not cause any changes in cell morphology ( Supplementary Figure 1) supports the choice of WR806 as host strain for SMLM imaging.…”

Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation

“…Strains and growth conditions. H. volcanii wild-type strain H53 and its derivatives (Table 1) were grown aerobically at 45℃ in liquid (orbital shaker at 250 rpm) or on solid semi-defined Hv-Cab medium (45). H53, ∆pibD, ∆pilA1-6, ∆pilB1C1, ∆pilB3C3, ∆pilB1C1B3C3, ∆arlA1, ∆arlA2, ∆arlA1-2, ∆aglB, and ∆agl15 media were additionally supplemented with tryptophan and uracil (both at 50 µg • ml -1 final concentration); cheB::tn, cheF::tn, ∆pssA, and ∆pssD media were supplemented with uracil (50 µg • ml -1 final concentration); H98 and ∆cetZ1 media were supplemented with thymidine and hypoxanthine (both at 40 µg • ml -1 final concentration) as well as uracil (50 µg • ml -1 final concentration) (46).…”

“…Solid media plates contained 1.5% (wt/vol) agar. Haloferax mediterranei was grown aerobically at 45℃ in Hv-Cab medium (45). Immersed liquid biofilm formation.…”

Cellular and genomic properties ofHaloferax gibbonsiiLR2-5, the host of euryarchaeal virus HFTV1

“…Hfx volcanii is reported to change its shape during growth in CA medium (Li et al, 2019;Silva et al, 2020). We analyzed the cell shape of LR2-5 in CA medium by phase contrast light microscopy.…”