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ABSTRACT Archaea, once thought limited to extreme environments, are now recognized as ubiquitous and fundamental players in global ecosystems. While morphologically similar to bacteria, they are a distinct domain of life and are evolutionarily closer to eukaryotes. The development of model archaeal systems has facilitated studies that have underscored unique physiological, biochemical, and genetic characteristics of archaea.Haloferax volcaniistands out as a model archaeon due to its ease of culturing, ability to grow on defined media, amenability to genetic and biochemical methods, as well as the support from a highly collaborative community. This haloarchaeon has been instrumental in exploring diverse aspects of archaeal biology, ranging from polyploidy, replication origins, and post-translational modifications to cell surface biogenesis, metabolism, and adaptation to high-salt environments. The extensive use ofHfx. volcaniifurther catalyzed the development of new technologies and databases, facilitating discovery-driven research that offers significant implications for biotechnology, biomedicine, and core biological questions. 

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. 

Abstract Archaea play indispensable roles in global biogeochemical cycles, yet many crucial 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 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 results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin’s cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains. 

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. Excluding archaea from high school and undergraduate curricula prevents students from learning the uniqueness and importance of this domain. Here, we have modified a familiar and popular microbiology experiment—the Kirby-Bauer disk diffusion antibiotic susceptibility test—to include, together with the model bacterium Escherichia coli , the model archaeon Haloferax volcanii . Students will learn the differences and similarities between archaea and bacteria by using antibiotics that target, for example, the bacterial peptidoglycan cell wall or the ribosome. Furthermore, the experiment provides a platform to reiterate basic cellular biology concepts that students may have previously discussed. We have developed two versions of this experiment, one designed for an undergraduate laboratory curriculum and the second, limited to H. volcanii , that high school students can perform in their classrooms. This nonpathogenic halophile can be cultured aerobically at ambient temperature in high-salt media, preventing contamination, making the experiment low-cost and safe for use in the high school setting. 

ABSTRACT 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 coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This m ulti p anel ad hesion (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 the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δ phz ) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation 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 an H. volcanii mutant that lacks archaella is 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. 

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N -glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N -glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx . volcanii N -glycoproteome revealed here provide new insights into the roles of N -glycosylation in archaeal cell biology. 

Abstract Motivation Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. Availability and implementation The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. Supplementary information Supplementary data are available at Bioinformatics online. 

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. 

ABSTRACT The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii . ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an Δ artA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the Δ hvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the Δ hvpssA and Δ hvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.