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.

Motility regulation plays a key role in prokaryotic responses to environmental stimuli. Here, we used a motility screen and selection to isolate hypermotile Haloferax volcanii mutants from a transposon insertion library. Whole genome sequencing revealed that hypermotile mutants were predominantly affected in two genes that encode HVO_1357 and HVO_2248. Alterations of these genes comprised not only transposon insertions but also secondary genome alterations. HVO_1357 contains a domain that was previously identified in the regulation of bacteriorhodopsin transcription, as well as other domains frequently found in two-component regulatory systems. The genes adjacent to hvo_1357 encode a sensor box histidine kinase and a response regulator, key players of a two-component regulatory system. None of the homologues of HVO_2248 have been characterized, nor does it contain any of the assigned InterPro domains. However, in a significant number of Haloferax species, the adjacent gene codes for a chemotaxis receptor/transducer. Our results provide a foundation for characterizing the root causes underlying Hfx. volcanii hypermotility.

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.

No abstract

Archaea, once thought to only live in extreme environments, are present in many ecosystems, including the human microbiome, and they play important roles ranging from nutrient cycling to bioremediation. Yet this domain is often overlooked in microbiology classes and rarely included in laboratory exercises.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δphz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δ phz ) form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δ phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that H. volcanii mutants that lack archaella are impaired in early stages of biofilm formation under shaking conditions. Importance: Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.