Identification and characterization of structural and regulatory cell-shape determinants inHaloferax volcanii

Abstract

Archaea play indispensable roles in global biogeochemical cycles, yet many critical cellular processes, including cell-shape determination, are poorly understood.Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify distinct mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The corresponding results indicated a diverse set of proteins, including transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. We also identified structural proteins, including a previously unknown cytoskeletal element, theHfx. volcaniiactin homolog volactin, which plays a role in disk-shape morphogenesis. In summary, we gleaned important insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

“…In summary, data presented here are consistent with the hy- TrmB family member TrmBL1 (Schiller et al, 2023). Further, glucose and other hexose sugars are precursors for N-glycosylation of pili involved in adhesion and subsequent biofilm formation (Esquivel et al, 2016;She et al, 2019)…”

“…In contrast, the distribution of cell shapes was significantly biased toward rod shape in ∆ tbsP and the double mutant, although the distribution remained heterogeneous. To further investigate ∆ tbsP cell shape under conditions that more heavily bias the parental strain toward disk shape (Schiller et al., 2023), ∆ tbsP cells were grown overnight to early stationary phase in medium lacking glucose and then visualized by microscopy. Under these conditions, ∆ tbsP cell morphology distribution remained clearly and significantly more rod‐like than that of the parent (Figure 5d, p < 2.2 × 10 −16 ), suggesting that TbsP is important for shifting from rod to disc shape in stationary phase.…”

“…Gene functions statistically overrepresented in each cluster are shown (p-values are cited in the text). Annotations for genes in each cluster, and whether the encoded proteins have been implicated in motility in previous studies (Esquivel et al, 2016;Schiller et al, 2023), are listed in Table S5. (c) Clusters with expression patterns consistent with TrmB-dependent induction and repression that are conserved with TrmB targets in other haloarchaeal species (Hackley et al, 2023, Schmid et al, 2009Todor et al, 2013Todor et al, , 2014.…”

“…To compare genes highlighted in our analysis with previously identified determinants of cell shape in Hfx. volcanii, we combined the lists of proteins exhibiting differential abundance between parental and cellshape defective strains (figures 2 and 4 of Schiller et al, 2023), resulting in 430 genes that were identified in at least one analysis (Table S5).…”

“…Rod‐shaped cells predominate at the motile leading edge of colonies swimming in soft agar, whereas disk‐shaped cells are found in the less motile center of motility colonies (Duggin et al., 2015). Genetic analyses have demonstrated that disk shape is associated with increased adhesion to abiotic surfaces, a proxy for biofilm formation (Legerme et al., 2016, Schiller et al., 2023). Conversely, rod shape is typically associated with increased motility.…”