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.

This first molecular biological study of archaeal immersed liquid biofilms advances our basic biological understanding of the model archaeon Haloferax volcanii. Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces.

The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. In the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation also play a role in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into Petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as chemotaxis transposon and aglB-deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the Petri-dish lid and are not dependent on changes in light, oxygen, or humidity. Taken together, this study demonstrates that H. volcanii requires novel, as yet unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements, providing the first evidence for a potential role for volatile signaling in H. volcanii.

Inducible promoters are one of the most important technical tools for cellular and molecular biology. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we develop and characterize a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions largely re-pressed in the absence of the above inducers. The most upregulated promoter was found to control the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two different 5' UTR fragments into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic region (P750). The P250 region showed to express msfGFP constitutively and its expression does not respond to the presence or ab-sence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP in the presence of the inducer. Finally, we validated our new inducible Pxyl promoter by reproducing morphological phenotypes al-ready described in the literature. By overexpressing the tubulin-like FtsZ1 or FtsZ2, we ob-served similar, but slightly more pronounced morphological defects compared to the trypto-phan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes with a new xylose-inducible promoter Pxyl that can be used simultaneously with the well-established PtnaA in functional studies in H. volcanii.