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1. Quorum sensing mediates morphology and motility transitions in the model archaeon Haloferax volcanii

   https://doi.org/10.1128/mbio.00906-25  

   Chatterjee, Priyanka; Consoli, Caroline E; Schiller, Heather; Winter, Kiersten K; McCallum, Monica E; Schulze, Stefan; Pohlschroder, Mechthild (June 2025, mBio)

   Newman, Dianne K (Ed.)

   ABSTRACT Quorum sensing (QS) is a population density-dependent mechanism of intercellular communication, whereby microbes secrete and detect signals to regulate behaviors such as virulence and biofilm formation. Although QS is well-studied in bacteria, little is known about cell-cell communication in archaea. The model archaeonHaloferax volcaniican transition from motile rod-shaped cells to non-motile disks as population density increases. In this report, we demonstrate that this transition is induced by a secreted small molecule present in cell-free conditioned medium (CM). The CM also elicits a response from a bacterial QS bioreporter, suggesting the potential for inter-domain crosstalk. To investigate theHfx. volcaniiQS response, we performed quantitative proteomics and detected significant differential abundances of 236 proteins in the presence of CM, including proteins involved in cell structure, motility, glycosylation, and two-component systems. We also demonstrate that a mutant lacking the cell shape regulatory factor DdfA does not undergo shape and motility transitions in the presence of CM, allowing us to identify protein abundance changes in the QS response pathway separate from those involved in shape and motility. In the ∆ddfAstrain, only 110 proteins had significant differential abundance, and comparative analysis of these two proteomics experiments enabled us to identify proteins dependent on and independent of DdfA in the QS response pathway. Our study provides the first detailed analysis of QS pathways in any archaeon, strengthening our understanding of archaeal communication as well as providing the framework for studying intra- and interdomain crosstalk. IMPORTANCEUnderstanding the complex signaling networks in microbial communities has led to many invaluable applications in medicine and industry. Yet, while archaea are ubiquitous and play key roles in nutrient cycling, little is known about the roles of archaeal intra- and interspecies cell-cell communication in environments such as the human, soil, and marine microbiomes. In this study, we established the first robust system for studying quorum sensing in archaea by using the model archaeonHaloferax volcanii. We demonstrated that different behaviors, such as cell shape and motility, are mediated by a signal molecule, and we uncovered key regulatory components of the signaling pathway. This work advances our understanding of microbial communication, shedding light on archaeal intra- and interdomain interactions, and contributes to a more complete picture of the interconnected networks of life on Earth. 

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2. Rapid rewiring of an archaeal transcription factor function via flexible cis–trans interactions

   https://doi.org/10.1091/mbc.E24-11-0505  

   Pastor, Mar Martinez; Darnell, Cynthia L; Vreugdenhil, Angie; Schmid, Amy K (July 2025, Molecular Biology of the Cell)

   Garner, Ethan (Ed.)

   For microbial cells, an appropriate response to changing environmental conditions is critical for viability. Transcription regulatory proteins, or transcription factors (TF) sense environmental signals to change gene expression. However, it remains unclear how TFs and their corresponding gene regulatory networks are selected over evolutionary time scales. The function of TFs and how they evolve are particularly understudied in archaeal organisms. Here, we identified, characterized, and compared the function of the RosR TF across three related hypersaline-adapted archaeal model species. RosR was previously characterized as a global regulator of gene expression during oxidative stress in the species Halobacterium salinarum ( hsRosR). Here, we use functional genomics and quantitative phenotyping to demonstrate that, despite strong sequence conservation of RosR across species, its function diverges substantially. Surprisingly, RosR in Haloferax volcanii ( hvRosR) and Haloferax mediterranei ( hmRosR) regulates genes whose products function in motility and the membrane, leading to significant defects in motility when RosR is deleted. Given weak conservation and degeneration in cis-regulatory sequences recognized by the RosR TF across species, we hypothesize that the RosR regulatory network is readily rewired during evolution across related species of archaea. 

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3. Haloferax volcanii Immersed Liquid Biofilms Develop Independently of Known Biofilm Machineries and Exhibit Rapid Honeycomb Pattern Formation

   https://doi.org/10.1128/mSphere.00976-20  

   Schiller, Heather; Schulze, Stefan; Mutan, Zuha; de Vaulx, Charlotte; Runcie, Catalina; Schwartz, Jessica; Rados, Theopi; Bisson Filho, Alexandre W.; Pohlschroder, Mechthild (December 2020, mSphere)

   Butler, Geraldine (Ed.)

   ABSTRACT 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. Additionally, in the model archaeon Haloferax volcanii , chemotaxis and AglB-dependent glycosylation play important roles 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 a chemotaxis-targeting 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, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements. IMPORTANCE 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. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms. 

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4. Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii reveals untranslated small RNAs with ribosome occupancy

   https://doi.org/10.1128/msphere.00343-25  

   Dallon, Emma; Moran, Haley M; Chidambaran, Sadhana R; Kian, Arman; Huang, Betty_Y H; Fried, Stephen D; DiRuggiero, Jocelyne (September 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized; however, little is known about the impacts of oxidative stress at the translation level. Using the model archaeonHaloferax volcanii, we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded by these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins inH. volcanii, revealed additional regulatory layers and underscored the multifaceted architecture of stress-responsive regulatory networks.IMPORTANCEArchaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. UsingHaloferax volcaniias a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation. 

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5. Mutations Affecting HVO_1357 or HVO_2248 Cause Hypermotility in Haloferax volcanii, Suggesting Roles in Motility Regulation

   https://doi.org/10.3390/genes12010058  

   Collins, Michiyah; Afolayan, Simisola; Igiraneza, Aime B.; Schiller, Heather; Krespan, Elise; Beiting, Daniel P.; Dyall-Smith, Mike; Pfeiffer, Friedhelm; Pohlschroder, Mechthild (January 2021, Genes)

   null (Ed.)

   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. 

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