Halobacterium salinarum are halophilic archaea that grow at or near saturating salt conditions. In addition to providing insight into how life copes with extreme conditions, studying Hbt. salinarum has been central to advances made in numerous fields, including bioenergetics, membrane protein structure determination and optogenetics. From the perspective of carbohydrate research, Hbt. salinarum provided the first example of N-glycosylation outside Eukarya. Yet, even 50 years after the first report of such post-translational modification in these haloarchaea, various aspects of Hbt. salinarum N-glycosylation seemingly unique to this organism remain largely unaddressed. These include questions related to the incorporation of iduronic acid in an N-linked glycan decorating Hbt. salinarum glycoproteins and the sulfation of this sugar at the O-3 position, as well as the transient methylation of this glycan at the lipid carrier- but not the protein-bound stage. In this review, recent progress on each of these unusual and unique aspects of Hbt. salinarum N-glycosylation is discussed.

The N-linked tetrasaccharide decorating glycoproteins of the halophilic archaea Halobacterium salinarum offered the first example of N-glycosylation outside the Eukarya and still represents the only known instance of iduronic acid (IdoA) being employed in this post-translational modification. Recent identification of Agl32 as the D-glucuronyl C5-epimerase catalyzing the conversion of glucuronic acid (GlcA) into IdoA allows for comparing this enzyme in each of the three domains of life, namely, Eukarya, Bacteria and Archaea. Specifically, the current study assessed whether Agl32 requires flanking sugars on either side of the target GlcA, as do its eukaryal and bacterial counterparts. Nuclear magnetic resonance analysis of the glycan from an Hbt. salinarum mutant unable to add the fourth and final N-linked tetrasaccharide sugar revealed that Agl32 requires GlcA on both sides of the target GlcA for the epimerization reaction. Despite similar requirements for flanking sugars, Agl32 shares little structural similarity with eukaryal GlcE or DSepi1, D-glucuronyl C5-epimerases respectively involved in IdoA generation in the glycosaminoglycans heparin/heparan sulfate and dermatan sulfate. Moreover, Agl32 processes a substrate far shorter than what is recognized by the eukaryal enzymes. Indeed, it would appear that Agl32 relies on a catalytic mechanism distinct from that employed by these other D-glucuronyl C5-epimerases. Finally, the presence of agl32 homologues in putative N-glycosylation gene clusters in other haloarchaea argues that the use of IdoA in N-glycosylation extends beyond Hbt. salinarum.

The crustacean exoskeleton features a micrometric, three-dimensional chitin scaffold. The intricate organization of this structure makes it an ideal model for investigating scaffold proteins at the nanoscale. Periodic exoskeleton replacement during a rapid and punctual molt cycle involves proteins that govern exoskeleton formation. Relying on binary expression pattern analysis of a molt-related transcriptomic library generated from the cuticle-forming epithelium of the crayfish Cherax quadricarinatus, a family of crustacean cuticle structural proteins termed 'crusticuls' was discovered and shown to present an exoskeleton formation-related expression pattern. All nine crusticuls include a chitin-binding domain bordered by two acidic residue-rich regions, putative functional domains related to exoskeletal formation and biomineralization. Crusticuls knock-down via RNAi resulted in over 95% reduced relative expression in treated versus control crayfish, with phenotypic effects ranging from prolonged molt cycles to lethality. Crusticuls were largely absent from newly formed cuticles following knockdown, resulting in exoskeletal deformities in the three-dimensional organization of chitinous bundles at the micro- and nanometric scales. These structural alterations were phenotypically translated into changes in cuticular hardness and elasticity. The identification of crusticuls as being key for proper nanometric three-dimensional organization of cuticular chitinous scaffolds opens new avenues for synthetic scaffold bio-mimetic applications.

In Halobacterium salinarum, halophilic archaea that grow in saturated salt solutions, glycoproteins are modified by an N-linked tetrasaccharide that includes iduronic acid (IdoA). This represents the only known example of IdoA, a sugar better known as a component of glycosaminoglycans found in various eukaryal tissues, in N-glycosylation. Although much of the pathway used to assemble this tetrasaccharide is defined, the epimerase that presumably converts glucuronic acid (GlcA) into IdoA had yet to be described. In silico predictions assign this role to VNG1058H. In Hbt. salinarum deleted of VNG1058H, an N-linked tetrasaccharide appeared in which IdoA is replaced by GlcA and greatly reduced sulfation of the last two tetrasaccharide sugars is noted. The absence of VNG1058H, moreover, affects cell physiology. Furthermore, Hbt. salinarum differentially transcribe VNG1058H as a function of growth temperature, suggestive of a response to environmental change. VNG1058H is the first D-glucuronyl C5-epimerase to be identified in Archaea.

ABSTRACT Despite providing the first example of archaeal N-glycosylation almost 50 years ago, detailed insight into the pathway used by Halobacterium salinarum to assemble and attach an N-linked tetrasaccharide decorating glycoproteins in this haloarchaea has only recently appeared. Still, numerous components of this pathway remain to be identified, including sulfotransferase(s), which modify the third and fourth tetrasaccharide sugars. In the present report, a series of bioinformatics, genetic, biochemical, and structural approaches served to reveal how membrane-associated VNG1056C and soluble VNG1057C respectively sulfate the iduronic acid at tetrasaccharide position three and the terminal glucuronic acid, seemingly independent of each other. Deletion of VNG1056C but not of VNG1057C reduced cell motility to a minor degree and did not cause archaellum filament bundling. Finally, transcription of VNG1056C or VNG1057C was augmented upon deletion of the other when cells were grown in low but not high salinity conditions possibly in an attempt to compensate for the loss of sugar sulfation resulting from the deletion. This augmented transcription, however, had no effect on the extent of tetrasaccharide sulfation. With demonstrated roles in Hbt. salinarum N-glycosylation, VNG1056C and VNG1057C were respectively re-annotated as Agl30 and Agl31, employing the nomenclature used to define archaeal N-glycosylation pathway components.IMPORTANCELike essentially all Archaea, the halophile Halobacterium salinarum performs N-glycosylation, namely, the covalent attachment of a glycan moiety to select asparagine residues in a target protein. Moreover, Hbt. salinarum represents one of the few current archaeal examples in which the pathway of N-glycosylation has been largely defined. Still, several components of this pathway remain to be defined, including the sulfotransferase(s) responsible for modifying the iduronic acid and glucuronic acid corresponding to the third and final sugars of the N-linked tetrasaccharide that decorates glycoproteins in this haloarchaeon. In the present report, a series of bioinformatics, genetic, biochemical, and structural approaches served to reveal how membrane-associated VNG1056C and soluble VNG1057C respectively sulfate the iduronic acid at tetrasaccharide position three and the terminal glucuronic acid, seemingly independent of each other. The need for two different enzymes reflects the sulfation of these sugars at distinct positions.

Many bacterial pathogens employ the type III secretion system (T3SS), a specialized complex that transports effector proteins that manipulate various cellular processes. The T3SS forms a translocon pore within the host-cell membrane consisting of two secreted proteins that transition from a soluble state into a transmembrane complex. Still, the exact sequence of events leading to the formation of a membranous functional pore remains uncertain. Here, we utilized the translocon proteins of enteropathogenic E. coli (EPEC) to investigate the sequence of those steps leading to translocon assembly, including self-oligomerization, hetero-oligomerization, interprotein interaction, and membrane insertion. We found that in EPEC, EspD (SctE) plays a dominant role in pore formation as it assembles into an oligomeric state, regardless of pH, membrane contact, or the presence of EspB (SctB). Subsequently, EspB subunits integrate into EspD homo-oligomers to create EspB-EspD hetero-oligomers that adopt a transmembrane orientation to create a functional pore complex.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

Protein degradation, which occurs in all cells, is essential for proper cellular function by regulating many cellular processes, destroying misfolded proteins, and providing protein building blocks under starvation conditions. As proteolysis is a destructive process, it is carried out by tightly regulated enzymes that evolved to interact with their protein substrates in a highly controlled and selective manner. The agents of protein degradation include proteasomes, AAA+ proteolytic machines found in all kingdoms of life. The bacterial proteasome specifically recognizes proteins conjugated to a protein tag termed Pup, with the proteasome regulatory particle, a ring-shaped hexamer termed Mpa in mycobacteria, being responsible for Pup recognition. Once Pup binds Mpa, Pup enters the central pore, where the Mpa AAA+ domain links ATP hydrolysis to the translocation of Pup and its conjugated substrate into a barrel-shaped proteasome core particle, where peptide bond cleavage occurs. As Pup traverses the Mpa pore en route to the AAA+ domain, it passes the inter-domain. Although the inter-domain is conserved in all proteasomes, its role in substrate processing remained unclear. We report here that the Mpa inter-domain promotes Pup binding via electrostatic interactions between conserved charged inter-domain pore loops and charged Pup residues. As such, the inter-domain serves as a gatekeeper that selects for Pup binding, thus facilitating tag interaction with the downstream AAA+ domain. Our findings thus reveal the existence of an additional level of substrate binding regulation in an AAA+ protease.

While performed by all three domains of life, N-glycosylation in Archaea is less well described than are the parallel eukaryal and bacterial processes. Still, what is known of the archaeal version of this universal post-translational modification reveals numerous seemingly domain-specific traits. Specifically, the biosynthesis of archaeal N-linked glycans relies on distinct pathway steps and components, rare sugars and sugar modifications, as well as unique lipid carriers upon which N-linked glycans are assembled. At the same time, Archaea possess the apparently unique ability to simultaneously modify their glycoproteins with very different N-linked glycans. In addition to these biochemical aspects of archaeal N-glycosylation, such post-translational modification has been found to serve a wide range of roles possibly unique to Archaea, including allowing these microorganisms to not only cope with the harsh physical conditions of the niches they can inhabit but also providing the ability to adapt to transient changes in such environments.

The swimming device of archaea—the archaellum—presents asparagine (N)-linked glycans. While N-glycosylation serves numerous roles in archaea, including enabling their survival in extreme environments, how this post-translational modification contributes to cell motility remains under-explored. Here, we report the cryo-EM structure of archaellum filaments from the haloarchaeon Halobacterium salinarum, where archaellins, the building blocks of the archaellum, are N-glycosylated, and the N-glycosylation pathway is well-resolved. We further determined structures of archaellum filaments from two N-glycosylation mutant strains that generate truncated glycans and analyzed their motility. While cells from the parent strain exhibited unidirectional motility, the N-glycosylation mutant strain cells swam in ever-changing directions within a limited area. Although these mutant strain cells presented archaellum filaments that were highly similar in architecture to those of the parent strain, N-linked glycan truncation greatly affected interactions between archaellum filaments, leading to dramatic clustering of both isolated and cell-attached filaments. We propose that the N-linked tetrasaccharides decorating archaellins act as physical spacers that minimize the archaellum filament aggregation that limits cell motility.

Halobacterium salinarum is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in Hbt. salinarum across a range of disciplines, including biophysics, chemistry, molecular evolu-tion and biotechnology. Hbt. salinarum have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on Hbt. salinarum, one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, Hbt. salinarum pre-sents creative solutions to the challenges of life in high salt.

In recent years, it has become clear that intrinsically disordered protein segments play diverse functional roles in many cellular processes, thus leading to a reassessment of the classical structure-function paradigm. One class of intrinsically disordered protein segments is entropic clocks, corresponding to unstructured random protein chains involved in timing cellular processes. Such clocks were shown to modulate ion channel processes underlying action potential generation, propagation, and transmission. In this review, we survey the role of entropic clocks in timing intra- and inter-molecular binding events of voltage-activated potassium channels involved in gating and clustering processes, respectively, and where both are known to occur according to a similar 'ball and chain' mechanism. We begin by delineating the thermodynamic and timing signatures of a 'ball and chain'-based binding mechanism involving entropic clocks, followed by a detailed analysis of the use of such a mechanism in the prototypical Shaker voltage-activated K+ channel model protein, with particular emphasis on ion channel clustering. We demonstrate how 'chain'-level alternative splicing of the Kv channel gene modulates entropic clock-based 'ball and chain' inactivation and clustering channel functions. As such, the Kv channel model system exemplifies how linkage between alternative splicing and intrinsic disorder enables the functional diversity underlying changes in electrical signaling.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

Flavin-dependent L-lactate dehydrogenase (LDH) from baker’s yeast (Saccharomyces cerevisiae) reversibly catalyzes the oxidation of L-lactate to L-pyruvate. In this study, four different enzymatic constructs were generated, and their catalytic and electrochemical properties were compared. Specifically, a truncated form of the native enzyme that includes only the catalytic domain, the native enzyme that includes an intrinsic electron-transferring cytochrome b2, a novel artificial enzyme containing a minimal cytochrome c and a version of the enzyme containing a fusion between two cytochromes were designed. All four variants were successfully expressed in Escherichia coli and presented properly matured heme domains. Assessing in vitro biocatalytic performance as reflected by lactate oxidation revealed the fusion-containing enzyme to be ∼ 12 times more active than the native enzyme. Electrochemical studies of electrode drop-casted enzyme variants also showed the superior performance of the dual-cytochrome construct, which displayed a lower average redox-potential for lactate oxidation, oxygen insensitivity in the lactate oxidation potential range and a wider dynamic range for lactate sensing, relative to the native enzyme. Moreover, product inhibition of this variant occurred at much higher lactate concentrations than with the native enzyme. In addition, when lower potentials were scanned using cyclic voltammetry, lactate-dependent oxygen reduction was measured for the dual-cytochrome fusion enzyme.

The attachment of a sugar to a hydrophobic lipid carrier is the first step in the biosynthesis of many glycoconjugates. In the halophilic archaeon Haloarcula hispanica, HAH_1206, renamed AepG, is a predicted glycosyltransferase belonging to the CAZy Group 2 family that shares a conserved amino acid sequence with dolichol phosphate mannose synthases. In this study, the function of AepG was investigated by genetic and biochemical approaches. We found that aepG deletion led to the disappearance of dolichol phosphate-glucuronic acid. Our biochemical assays revealed that recombinant cellulose-binding, domain-tagged AepG could catalyze the formation of dolichol phosphate-glucuronic acid in time-and dose -dependent manners. Based on the in vivo and in vitro analyses, AepG was confirmed to be a dolichol phosphate glucuronosyltransferase involved in the synthesis of the acidic exopolysaccharide produced by H. hispanica. Furthermore, lack of aepG resulted in hindered growth and cell aggregation in high salt medium, indicating that AepG is vital for the adaptation of H. hispanica to a high salt environment. In conclusion, AepG is the first dolichol phosphate glucur-onosyltransferase identified in any of the three domains of life and, moreover, offers a starting point for further investigation into the diverse pathways used for extracellular polysaccharide biosynthesis in archaea.

N-glycosylation is a post-translational modification of proteins that occurs across all three domains of life. In Archaea, N-glycosylation is crucial for cell stability and motility, but importantly also has significant implications for virus–host interactions. While some archaeal viruses present glycosylated proteins or interact with glycosylated host proteins, the direct influence of N-glycosylation on archaeal virus–host interactions remains to be elucidated. In this study, we generated an N-glycosylation-deficient mutant of Halorubrum lacusprofundi, a halophilic archaeon commonly used to study cold adaptation, and examined the impact of compromised N-glycosylation on the infection dynamics of two very diverse viruses. While compromised N-glycosylation had no influence on the life cycle of the head-tailed virus HRTV-DL1, we observed a significant effect on membrane-containing virus HFPV-1. Both intracellular genome numbers and extracellular virus particle numbers of HFPV-1 were increased in the mutant strain, which we attribute to instability of the surface-layer which builds the protein envelope of the cell. When testing the impact of compromised N-glycosylation on the life cycle of plasmid vesicles, specialized membrane vesicles that transfer a plasmid between host cells, we determined that plasmid vesicle stability is strongly dependent on the host glycosylation machinery. Our study thus provides important insight into the role of N-glycosylation in virus–host interactions in Archaea, while pointing to how this influence strongly differs amongst various viruses and virus-like elements.

Archaea are microorganisms that comprise a distinct branch of the universal tree of life and which are best known as extremophiles, residing in a variety of environments characterized by harsh physical conditions. One seemingly universal trait of Archaea is the ability to perform N-glycosylation. At the same time, archaeal N-linked glycans present variety in terms of both composition and architecture not seen in the parallel eukaryal or bacterial processes. In this mini-review, many of the unique and unusual sugars found in archaeal N-linked glycans as identified by nuclear magnetic resonance spectroscopy are described.

Halobacterium salinarum, a halophilic archaeon that grows at near-saturating salt concentrations, provided the first example of N-glycosylation outside Eukarya. Yet, almost 50 years later, numerous aspects of such post-translational protein processing in this microorganism remain to be determined, including the architecture of glycoprotein-bound glycans. In the present report, nuclear magnetic resonance spectroscopy was used to define a tetrasaccharide N-linked to both archaellins, building blocks of the archaeal swimming device (the archaellum), and the S-layer glycoprotein that comprises the protein shell surrounding the Hbt. salinarum cell as β-GlcA(2S)-(1 → 4)-α-IdoA(3S)-(1 → 4)-β-GlcA-(1 → 4)-β-Glc-Asn. The structure of this tetrasaccharide fills gaps remaining from previous studies, including confirmation of the first known inclusion of iduronic acid in an archaeal N-linked glycan. At the same time, the sulfation of this iduronic acid at the O-3 position has not, to the best of our knowledge, been previously seen. As such, this may represent yet another unique facet of N-glycosylation in Archaea.

In the archaeon Haloferax volcanii , the dolichol phosphate (DolP)-mannose synthase AglD charges the lipid DolP with mannose, which is delivered to a protein-bound tetrasaccharide to generate the pentasaccharide decorating glycoproteins in this organism. Structural studies demonstrated the similarity of AglD to Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase with a solved three-dimensional structure.