Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea.

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.

SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides.

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.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

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 multipanel 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 the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) 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 Δphz 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.

Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal.

The identification of peptide sequences and their post-translational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs). Whereas the benefits of combining results from multiple protein database search engines have been previously established, similar approaches for OMS results have been missing so far. Here we compare and combine results from three different OMS engines, demonstrating an increase in peptide spectrum matches of 8-18%. The unification of search results furthermore allows for the combined downstream processing of search results, including the mapping to potential PTMs. Finally, we test for the ability of OMS engines to identify glycosylated peptides. The implementation of these engines in the Python framework Ursgal facilitates the straightforward application of the OMS with unified parameters and results files, thereby enabling yet unmatched high-throughput, large-scale data analysis.

Improved growth and morphological plasticity of Haloferax volcanii.

Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped ('plate') cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace element micronutrients. With these supplemented media, transient development of plate cells into uniformly shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies ('pleomorphic rods') were observed at all growth stages of the culture; the highly elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early log-phase rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results reveal the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey.

Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.

Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non-homologous sortases from archaea and bacteria.

Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in Haloferax volcanii depends upon the peptidase archaeosortase A (ArtA) processing C-termini of substrates containing C-terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid-modified C-terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram-positive bacteria, which also process C-termini of substrates whose C-terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His-Cys-Arg residues of sortase catalytic sites. The study presented here shows that ArtAWT -GFP expressed in trans complements ΔartA growth and motility phenotypes, while alanine substitution mutants, Cys173 (C173A), Arg214 (R214A) or Arg253 (R253A), and the serine substitution mutant for Cys173 (C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtAC173A -GFP, ArtAC173S -GFP and ArtAR214A -GFP cannot process substrates, while replacement of the third residue, ArtAR253A -GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.

ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus.

Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of Haloferax volcanii, previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, in silico analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved "twin" arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry.IMPORTANCE The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon H. volcanii, through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for in silico subcellular localization predictions of archaeal and bacterial proteins.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation.

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.

Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

UNLABELLED: For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. IMPORTANCE: Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems.

A conserved type IV pilin signal peptide H-domain is critical for the post-translational regulation of flagella-dependent motility.

In many bacteria and archaea, type IV pili facilitate surface adhesion, the initial step in biofilm formation. Haloferax volcanii has a specific set of adhesion pilins (PilA1-A6) that, although diverse, contain an absolutely conserved signal peptide hydrophobic (H) domain. Data presented here demonstrate that these pilins (PilA1-A6) also play an important role in regulating flagella-dependent motility, which allows cells to rapidly transition between planktonic and sessile states. Cells lacking adhesion pilins exhibit a severe motility defect, however, expression of any one of the adhesion pilins in trans can rescue the motility and adhesion. Conversely, while deleting pilB3-C3, genes required for PilA pilus biosynthesis, results in cells lacking pili and having an adhesion defect, it does not affect motility, indicating that motility regulation requires the presence of pilins, but not assembled pili. Mutagenesis studies revealed that the pilin-dependent motility regulatory mechanism does not require the diverse C-terminal region of the PilA pilins but specifically involves the conserved H-domain. This novel post-translational regulatory mechanism, which employs components that promote biofilm formation to inhibit motility, can provide a rapid response to changing environmental conditions. A model for this regulatory mechanism, which may also be present in other prokaryotes, is discussed.

Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii, and its use for gene discovery.

BACKGROUND: Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. RESULTS: Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. CONCLUSIONS: We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.

Haloferax volcanii archaeosortase is required for motility, mating, and C-terminal processing of the S-layer glycoprotein.

Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S-layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase-like system for proteolysis-coupled, carboxy-terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low-salt conditions, (b) alterations in cell shape and the S-layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S-layer glycoprotein, using detailed proteomic analysis. While the carboxy-terminal region of S-layer glycoproteins, consisting of a putative threonine-rich O-glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild-type strain. This clearly shows that ArtA is involved in carboxy-terminal post-translational processing of the S-layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy-terminal region of the S-layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis-coupled, covalent cell surface attachment.

Identification of structural and regulatory cell-shape determinants in Haloferax volcanii.

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.

Novel archaeal adhesion pilins with a conserved N terminus.

Type IV pili play important roles in a wide array of processes, including surface adhesion and twitching motility. Although archaeal genomes encode a diverse set of type IV pilus subunits, the functions for most remain unknown. We have now characterized six Haloferax volcanii pilins, PilA[1-6], each containing an identical 30-amino-acid N-terminal hydrophobic motif that is part of a larger highly conserved domain of unknown function (Duf1628). Deletion mutants lacking up to five of the six pilin genes display no significant adhesion defects; however, H. volcanii lacking all six pilins (ΔpilA[1-6]) does not adhere to glass or plastic. Consistent with these results, the expression of any one of these pilins in trans is sufficient to produce functional pili in the ΔpilA[1-6] strain. PilA1His and PilA2His only partially rescue this phenotype, whereas ΔpilA[1-6] strains expressing PilA3His or PilA4His adhere even more strongly than the parental strain. Most surprisingly, expressing either PilA5His or PilA6His in the ΔpilA[1-6] strain results in microcolony formation. A hybrid protein in which the conserved N terminus of the mature PilA1His is replaced with the corresponding N domain of FlgA1 is processed by the prepilin peptidase, but it does not assemble functional pili, leading us to conclude that Duf1628 can be annotated as the N terminus of archaeal PilA adhesion pilins. Finally, the pilin prediction program, FlaFind, which was trained primarily on archaeal flagellin sequences, was successfully refined to more accurately predict pilins based on the in vivo verification of PilA[1-6].

Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

N-glycosylation of Haloferax volcanii flagellins requires known Agl proteins and is essential for biosynthesis of stable flagella.

N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

Proteomic Sample Preparation and Data Analysis in Line with the Archaeal Proteome Project.

Despite the ecological, evolutionary and economical significance of archaea, key aspects of their cell biology, metabolic pathways, and adaptations to a wide spectrum of environmental conditions, remain to be elucidated. Proteomics allows for the system-wide analysis of proteins, their changes in abundance between different conditions, as well as their post-translational modifications, providing detailed insights into the function of proteins and archaeal cell biology. In this chapter, we describe a sample preparation and mass spectrometric analysis workflow that has been designed for Haloferax volcanii but can be applied to a broad range of archaeal species. Furthermore, proteomics experiments provide a wealth of data that is invaluable to various disciplines. Therefore, we previously initiated the Archaeal Proteome Project (ArcPP), a community project that combines the analysis of multiple datasets with expert knowledge in various fields of archaeal research. The corresponding bioinformatic analysis, allowing for the integration of new proteomics data into the ArcPP, as well as the interactive exploration of ArcPP results is also presented here. In combination, these protocols facilitate an optimized, detailed and collaborative approach to archaeal proteomics.

Immersed Liquid Biofilm and Honeycomb Pattern Formations in Haloferax volcanii.

Biofilms are cellular aggregates encased in extracellular polymeric substances and are commonly formed by single-celled eukaryotes, bacteria, and archaea. In addition to attaching to solid surfaces, these cellular aggregates can also be observed floating on or immersed within liquid cultures. While biofilms on surfaces have been studied in some archaea, little is known about liquid biofilms. Surprisingly, immersed liquid biofilms of the model archaeon Haloferax volcanii do not require the same set of machinery needed to form surface-attached biofilms. In fact, to date not a single gene has been identified that is involved in forming immersed liquid biofilms. Interestingly, after an immersed liquid biofilm forms, removal of the Petri dish lid induces rapid, transient, and reproducible honeycomb patterns within the immersed liquid biofilm itself, triggered by a reduction in humidity. In this chapter, we outline a protocol for both immersed liquid biofilm and honeycomb pattern formations. This protocol will be essential for determining the novel components required for the formation of immersed liquid biofilms and honeycomb patterns.

Accessible and Insightful Scientific Learning Experiences Using the Microorganism Haloferax volcanii.

Early exposure to science is critical to incite interest in scientific careers, promote equity and retention in STEM fields, and increase the general understanding of the scientific method. For many educators, however, the myriad resources that many scientific experiments require are not readily available. Microbiology experiments in particular can often be inaccessible for a lot of classrooms. In addition, microbiological studies often involve eukaryotic microbes and bacteria while excluding an entire domain of life: archaea. Archaea are more closely related to eukaryotes than are bacteria, and although all prokaryotic cells lack a nucleus, various key aspects of the cell biology of archaea and bacteria are fundamentally different. In addition to being useful for teaching about the diversity and evolution of living organisms, these differences between archaea and bacteria can also be harnessed to teach and emphasize other important biological topics. Haloferax volcanii is a non-pathogenic model haloarchaeon that allows for safe, affordable, and accessible microbiological experiments, as the requirement of high-salt media to grow H. volcanii presents a low risk of contamination. Here, we describe how H. volcanii can be used in the classroom and outline a protocol demonstrating their resistance to a broad spectrum of antibiotics, underscoring the distinct cell biology of bacteria and archaea. Finally, we introduce strategies and protocols to perform this and other H. volcanii experiments such that they can be performed based on the resources available in a high school or undergraduate classroom.