Sweet New Roles for Protein Glycosylation in Prokaryotes

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Review

Sweet New Roles for Protein Glycosylation in Prokaryotes Jerry Eichler1,* and Michael Koomey2 Long-held to be a post-translational modification unique to Eukarya, it is now clear that both Bacteria and Archaea also perform protein glycosylation, namely the covalent attachment of mono- to polysaccharides to specific protein targets. At the same time, many of the roles assigned to this proteinprocessing event in eukaryotes, such as guiding protein folding/quality control, intracellular trafficking, dictating cellular recognition events and others, do not apply or are even irrelevant to prokaryotes. As such, protein glycosylation must serve novel functions in Bacteria and Archaea. Recent efforts have begun to elucidate some of these prokaryote-specific roles, which are addressed in this review.

Trends Because many of the roles assumed by protein glycosylation in eukaryotes are not applicable to Bacteria or Archaea, this postmodification likely serves distinct roles in prokaryotes. In Bacteria, protein glycosylation systems are found in nonpathogenic species, pointing to roles beyond virulence. In Bacteria and Archaea, protein glycosylation contributes to the integrity and proper architecture of glycoprotein-containing assemblies.

Prokaryotic Protein Glycosylation – Understanding the How but Not the Why As the list of completed genome sequences keeps growing, it is becoming increasingly clear that the number of protein-coding genes cannot alone account for the size of an organism’s proteome. Sources of proteomic expansion include the various post-translational modifications (see Glossary) a given protein can undergo. Of the various protein-processing events that have been described, glycosylation, namely the covalent linkage of mono- to polysaccharides, is one of the most prevalent and probably the most complex (Box 1) [1,2]. Long thought to be restricted to Eukarya, it is now accepted that both Bacteria and Archaea also are capable of N-glycosylation, where glycans are amide-bonded to select Asn residues of a target protein, as well as O-glycosylation, where glycans are added to hydroxyl-presenting amino acids, particularly Ser and Thr [3–6] (Table 1). Despite the fact that numerous glycoproteins have been identified in Bacteria and Archaea [7,8], that the structures of many of the glycans decorating prokaryal glycoproteins have been solved [9–13], and that considerable progress has been made in delineating pathways of protein glycosylation in several bacterial and archaeal species [6,14,15], the roles served by the bacterial and archaeal versions of this universal post-translational modification remain poorly defined. In Eukarya, numerous functions have been assigned to protein glycosylation. The glycosylation process begins in the endoplasmic reticulum, the first stop on the secretory pathway, where a lipid-bound polysaccharide core is transferred to target protein Asn residues. The N-linked glycan is then augmented by individual sugars, also transferred from lipid carriers, to yield a complex branched oligosaccharide [16–18]. The composition of the N-linked glycan dictates interactions of the modified protein with molecular chaperones, such as calnexin and calreticulin, and other enzymes that accommodate proper protein folding [19,20]. Indeed, thermodynamics-based studies have demonstrated the importance of protein glycosylation for protein folding [21,22]. At the same time, the same N-linked glycan structure is monitored by the quality-control system responsible for identifying aberrantly folded proteins and targeting them for degradation, if necessary [23–25]. Once an N-glycosylated protein has successfully navigated the coordinated protein [203_TD$IF]folding and quality control steps, it may be delivered to the Golgi, the next station along the secretory pathway, via a sorting process that can also rely on N-linked

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Changes in protein glycosylation offers prokaryotes a rapid and reversible manner in which to respond to environmental changes.

1

Department of Life Sciences, Ben Gurion University of the Negev, Beersheva 84105, Israel 2 Department of Biosciences, University of Oslo, 0316 Oslo, Norway

*Correspondence: [email protected] (J. Eichler).

http://dx.doi.org/10.1016/j.tim.2017.03.001 © 2017 Elsevier Ltd. All rights reserved.

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Box 1. Glycosylation [201_TD$IF]As a Source of Protein Diversity

Glossary

Of the different post-translation modifications to which a given protein can be subjected, glycosylation introduces the most diversity. Several factors are responsible for the enormous variability associated with protein glycosylation. In addition to the variability derived from how a glycan is linked to a protein (e.g., N-linked or O-linked), considerable diversity is generated at the level of individual sugars comprising protein-linked glycans. For instance, the incorporation of sugars that differ in the number of backbone carbons (e.g., pentoses and hexoses), that can exist in different epimeric forms (e.g., glucose, mannose and galactose), and that can be distinguished via the addition of different chemical groups (e.g., amino or methyl groups) all contribute to glycan diversity. Further variablity arises when sugars start to oligomerize into a glycan due to the many possible linkages between any two sugars (in terms of both the position and stereochemistry of the connection), the possibility for branching, and the heterogeneity possible in a given oligosaccharide. Indeed, the variability of protein-linked glycans may be infinite because of the fact that no template limiting the size of an oligosaccharide seems to exist. Together, these considerations result in a plethora of protein-linked glycans unique in composition and/or architecture.

Archaellum: the motility structure of Archaea, functionally equivalent to the bacterial flagellum. Autotransporters: found in a broad range of Gram-negative bacteria, autotransporters comprise a family of outer membrane or secreted proteins that facilitate their own transport to the cell surface. In such proteins, the autotransporter domain, comprising the C-terminal portion of the protein, forms a beta-barrel structure in the outer membrane through which the N-terminal domain is presented on the cell surface. Autotransporters are associated with virulence, contributing to adhesion, aggregation, invasion, biofilm formation, and toxicity. N-glycosylation: the covalent linkage of glycans to select Asn residues in a target protein through an amide bond. O-glycosylation: the covalent linkage of glycans to hydroxylpresenting amino acids, particularly Ser and Thr. Oligosaccharyltransferase: oligosaccharyltransferases catalyze the transfer of glycans from the lipid carriers upon which they are assembled onto selected residues in glycoproteins. In Bacteria, Archaea, and lower eukaryotes, the oligosaccharyltransferase acts alone, whereas in higher Eukarya, the enzyme exists as a multimeric complex. Post-translation modification: an event that follows translation, designed to create variants of a given protein through the covalent attachment of one or more of several classes of molecules (e.g., sugars, lipids, or small chemical groups, like acetyl or methyl groups), the formation of intra- or intermolecular linkages (e.g., disulfide bonds), proteolytic cleavage (e.g., signal peptide removal), and/or any combination thereof. Sequon: sequence motifs in a polypeptide denoting sites where glycans are attached. In Nglycosylation, the Asn of a sequon, namely an Asn-Xaa-Ser/Thr sequence, where Xaa is any residue but Pro, is modified. Variations to the canonical sequon have been observed in prokaryotes. The sequons processed in Oglycosylation are less well defined.

glycan composition [26,27]. Once in the Golgi, the N-linked glycan is subjected to further processing through the addition and/or removal of constituent sugars to yield a range of Nlinked oligosaccharides [28–30]. The Golgi is also the site of O-glycosylation, a second major protein glycosylation event that can also introduce considerable diversity into the glycosylation profile of a glycoprotein [31,32]. In functional terms, the heterogeneity in glycan content generated in the Golgi can be exploited for targeting different glycoproteins to distinct subcellular compartments [33,34] or, in the case of cell-surface-exposed glycoproteins, can contribute to various cell–cell or other recognition events important for the development, differentiation, or physiology of a particular cell, tissue, or organism [35–38]. Such hetereogeneity can, moreover, reflect different diseased states [39–42]. At the same time, it would appear that many of the roles assumed by protein glycosylation in eukaryotes are not relevant for prokaryotes. For instance, whereas protein folding and Nglycosylation are linked in the eukaryal secretory pathway, these processes occur on either side of the plasma membrane in the case of bacterial and archaeal proteins secreted by the twin arginine translocation pathway. Here, such proteins fold in the cytoplasm [43,44], while oligosaccharyltransferase-based N-glycosylation transpires on the outer surface of the bacterial and archaeal cell [45,46] (as do some versions of bacterial O-glycosylation [47]). Likewise, the need to sort proteins to distinct subcellular compartments is extremely limited in prokaryotes. Finally, the number of recognition events required by a prokaryotic cell is likely to be far less than its eukaryal counterpart. Therefore, Bacteria and Archaea must rely on protein glycosylation for other purposes than how this [204_TD$IF]post-translational modification is used in Eukarya (Figure 1, Key Figure). In this review, recent works addressing these roles are discussed.

Bacterial Protein Glycosylation – Not for Virulence Alone The number of bacterial protein glycosylation systems recognized continues to grow. This process has been fueled in part by a few serendipitous discoveries followed by comparative genomics that allows one to immediately see how broadly distributed protein glycosylation systems (and genes) truly are. The take-home messages here are that (i) bacterial protein glycosylation is much more prevalent than one could have imagined, and (ii) it is not strictly associated with pathogenic species. There are thus a number of outstanding questions relating to the biological significance of bacterial protein glycosylation that are emphasized here. In studying protein glycosylation in Bacteria, the predominant emphasis has been placed on pathogens. However, systems related to those found in pathogens abound in commensal and environmental isolates. Thus, bacterial protein glycosylation is not a canonical virulence factor as defined by the criteria established by Falkow [48]. Nonetheless, clear defects in colonization and virulence in mammalian, insect, and plant model systems are seen for glycosylation null mutants. This is particularly true of so-called dedicated systems in which the glycosylation

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S-layer: part of the cell envelope, the surface layer is a self-assembling two-dimensional pseudocrystalline array that covers the entire outer surface of the cell. Comprising a single or a very small number of proteins or glycoproteins, the S-layer is ubiquitous in Archaea and is common in Bacteria.

Table 1. N- and O-glycosylation in Eukarya, Bacteria, and Archaea Eukarya

Bacteria

Archaea

Essential

Yes

No

Some species

Distribution

Universal

Limited

Almost universal

Glycan diversity

Common core

Limited

Extensive

Glycan lipid carrier

Dolichol phosphate, dolichol pyrophosphate

Undecaprenol pyrophosphate

Dolichol phosphate, dolichol pyrophosphate

[20_TD$IF]Number of subunits

Single or multiple

Single

Single

Catalytic subunit

STT3

PglB

AglB

Processing of proteinbound glycan

Yes

No

Yes

Different glycans on same protein

No

No

Yes

Universal

Some species

Unknown

N-glycosylation

Oligosaccharyltranferase

O-glycosylation Distribution Glycan diversity

Extensive

Extensive

Limited

Transfer of assembled glycan from lipid carrier

No

Possible

Unknown

Processing of proteinbound glycan

Yes

No

Unknown

machineries target a specific protein. These most often involve major surface-localized entities, such as flagella, autotransporters, and type IV pili. Bacterial flagella come in both O-glycosylated and non-glycosylated forms [49]. In those cases where flagellin subunits undergo modification, null glycosylation mutants show a remarkable array of disparate phenotypes. In Helicobacter, Campylobacter, and Aeromonas, as well as Gram-positive Clostridia and Listeria, glycosylation-null mutants are nonflagellated and display fla
[20_TD$IF] phenotypes [50–54]. In contrast, such mutants in Pseudomonas and Burkholderia retain flagella and motility [55,56]. A breakthrough in understanding how flagellin glycosylation might exert its influence on filament assembly came from studies in Aeromonas caviae. Using a mutant lacking the Maf1 glycosyltransferase required for the transfer of pseudaminic acid to flagellin, Parker and colleagues established that glycosylation is dispensable for subunit export but is essential for filament assembly, since nonglycosylated flagellin is still secreted [57]. Flagellin glycosylation in a chaperone mutant (flaJ) showed that glycosylation takes place in the cytoplasm (as long surmised but never previously documented) and before chaperone binding and protein secretion. It was further shown that FlaJ preferentially bound glycosylated flagellin. It will be interesting and important to see if such findings can be extended to encompass the other flagellar glycosylation systems. Bacterial autotransporters, adhesins of Gram-negative bacteria defined by shared structural elements and surface localization mechanisms, also come in both glycosylated and nonglycosylated flavors. O-glycosylated autotransporters are modified with heptoses at multiple sites within the passenger domain via the action of cytoplasmic heptosyltransferases using ADP-heptose [58]. Three such adhesins have been characterized in Escherichia coli, namely AIDA-1, Ag43, and TibA. Surprisingly, glycosylation null mutants have different effects on otherwise shared phenotypes but no major quantitative effects on secretion or surface

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Key Figure

Protein Glycosylation Serves Distinct Roles in Eukarya and in Bacteria and Archaea

Eukarya

Protein folding and quality control

Cell–cell and other recognion events

Differenal subcellular targeng

Protein glycosylaon

Bacteria Archaea

Assembly and strength of glycoproteinbased structures

Remodeling in response to environmental changes

Virulence, host–microbe interacons

Figure 1. Given differences in the temporal relationship and location of protein glycosylation, in the structure of the cell, in interactions with biological molecules or other cells, and in lifestyle, eukaryotes and prokaryotes (Bacteria and Archaea) rely on protein glycosylation for distinct purposes.

localization. Such mutants in the Ag43 system were found to be defective in associated functions, such as adherence and autoagglutination, yet showed enhanced binding to elements of the extracellular matrix [59]. In contrast, heptosyltransferase null mutants in the TibA system were altered solely in adherence phenotype [60]. These findings are consistant with others, revealing how poorly understood the structure–function relationships of autotransporters are with regard to their different phenotypes. The recent, highly resolved structure of a glycosylated 300 residue-long TibA peptide represents a major advance in this arena [61]. That same study also defined a unique family of structural autotransporter heptosyltransferases and determined their structure. Another breakthrough in this field was the identification of an entirely different class of heptosyltransferases mediating N-linked glycosylation of a subset of autotransporters in species including Actinobacillus, Kingella, and Aggregatibacter [62,63]. Here, the cytosolic N-glycosyltransferase utilizes nucleotide-activated monosaccharides to modify asparagine residues with a relaxed consensus sequon. These [205_TD$IF]enzymes are related to HMW1C that Nglycosylates adhesins in Haemophilus influenzae [64,65]. Null mutants for the glycosyltransferase are functionally defective for autotransporter function, as are the equivalent mutants in the Olinked systems. However, it remains unclear whether these phenotypes result from reduced function per se, as opposed to a reduction in surface localization. It is remarkable that despite the fact that the Aggregatibacter aphrophilus autotransporter is almost identical to that in Aggregatibacter actinomycetemcomitans, the former is N-glycosylated while the latter is O-glycosylated in a process involving enzymes used in the biosynthesis of the O-polysaccharide of lipopolysaccharide, including the WaaL-type O-antigen ligase [66]. Moreover, an HMW1C-like N-glycosyltransferase is not readily identifiable in the A. actinomycetemcomitans genome. N- and O-linked broad-spectrum (aka general) protein glycosylation systems encompass those that modify a large number of extracytoplasmically targeted substrates and are exemplified by the N-linked system of Campylobacter jejuni and the O-linked systems of pathogenic Neisseria sp. and Mycobacterium tuberculosis, respectively. In C. jejuni, substrates are modified with en

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bloc transferred oligosaccharides by an oligosaccharyltransferase structurally related to the STT3 subunit of the eukaryotic oligosaccharyltransferase complex [67,68]. The Neisseria sp. system involves the en bloc transfer of oligosaccharides by an oligosaccharyltransferase structurally related to the WaaL family of O-antigen ligases [15,69], while the mycobacterial systems rely on the transfer of mannose from polyprenol carriers by protein mannosyltransferases related to their eukaryotic counterparts [70,71]. Despite their differences, a number of shared themes connect these systems. First, membrane-linked protein substrates (lipoproteins or proteins bearing membrane-spanning domains) are over-represented in all three. Likewise, the vast majority of glycoproteins in each are not predicted to be surface-exposed. In the C. jejuni and Neisseria systems, each utilizes a di-N-acetyl-bacillosamine sugar at the reducing end of the glycan that is synthesized and transferred to undecaprenylphosphate by a conserved and evolutionarily related set of components [72,73]. Although the Neisseria and mycobacterial systems do not utilize classical targeting sequons, the sites of modified Ser and Thr residues in each lie within interdomain regions of reduced complexity enriched in proline, as well as threonine and serine residues. Taken together, these systems exemplify how amalgamations of related components connect, which otherwise are perceived as being highly unrelated. Broad-spectrum systems have also been identified in Acinetobacter and Burkholderia via bioinformatics, and each of these also encompass glycosylation of the pilin subunit protein of the type IV pilus colonization factor [74,75]. Again, mutants defective in glycosylation are attenuated in infection models for both. It should be noted as a matter of curiosity that an active protein-targeting oligosaccharyltransferase (VC0393) has been identified in Vibrio cholerae that O-glycosylates target proteins from Neisseria when coexpressed in Escherichia coli [76]. However, protein glycosylation has yet to be observed in V. cholerae. In addition, a forward genetic screen using Tn-seq showed no fitness alterations during infection, dissemination, or survival in the aquatic environment for the VC0393 null mutant [77]. Future work needs to resolve the status of a potential V. cholerae protein O-glycosylation system. Nearly all of these systems are associated with pathogens, and glycosylation null mutants have been seen to be colonization-defective or of reduced virulence in host model systems (often while showing few, if any, in vitro phenotypes) [74,78–81]. Understanding the mechanisms behind these attenuated phenotypes is complicated by the large number of proteins lacking glycosylation. Thus, it remains unclear whether these phenotypes are the result of the nonglycosylation of a single protein or the cumulative effects of multiple protein [206_TD$IF]perturbations. As noted earlier, similar glycosylation systems are found in related commensal and environmental species in each of the above cases. The unique general O-linked system documented in Bacteroides fragilis is particularly interesting as this species is considered to be an important member of the healthy gut microbiota [82]. Mutants with altered glycan structures were unable to compete with wild-type organisms in gut colonization. In addition, this seems to be unique among broad-spectrum O-glycosylation systems in its use of a sequon-like element. Although the oligosaccharyltransferase has yet to be defined in this system, the basic structure of the glycoforms (and some of the biosynthetic machinery), as well as a defined glycoproteome, has been established. Little attention is placed here on the detailed synthetic pathways and precise structures of the glycans/oligosaccharides themselves. Needless to say, there is an enormous amount of diversity to be found. That said, one can ask why specific glycoforms are associated with specific systems. Might they be driven by forces of the innate and/or adaptive immune systems? Or are they shaped by mere metabolic considerations relating to the costs of synthesizing certain oligosaccharides or conflicts with coexpressed glycoconjugate synthesis pathways? In some cases, the systems merely tap into or co-opt a pre-existing pathway for

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lipopolysaccharide or capsular polysaccharide biosynthesis. It is conceivable that such glycans might mimic host glycan structure and thus perturb or ameliorate immune recognition or, more generally, merely mask elements that might otherwise elicit immune responses [83]. In some bacteria, glycoform structure is very stable, while in others there are significant degrees of intraand interstrain diversity. This situation is perhaps exemplified by the related C. jejuni and pathogenic Neisseria broad-spectrum systems. The former utilizes an incredibly conserved heptasaccharide, while the latter employs an array of glycoforms that can be expressed by a single strain. Why is the system so constrained in the case of the former and so deconstrained in the latter? Likewise, some flagellin systems are very static while in others, such as those of Helicobacter, Campylobacter, and Clostridium species, large genomic islands associated with dramatic glycoform variability are found. With regard to the adaptive immune response, it is notable that only few studies have examined antibody responses to protein-associated glycans or the effects of glycan-specific antibodies. This is surprising, first as it is well established that conjugating oligosaccharides to a protein results in transforming such oligosaccharides into Tcell-dependent antigens, underlying a robust antibody response. Second, many glycoengineering endeavors are predicated on exploiting bacterial protein glycosylation systems to produce vaccines targeting bacterial glycoconjugates (Box 2). Two recent works suggest that glycan-specific antibodies may have significant biological activities. Of note, Pluschke and colleagues identified a Bacillus anthracis lineage lacking expression of the spore surface-linked oligosaccharide and suggested that this could be the result of prior immunization of cattle with a vaccine based on oligosaccharide-positive spores [84]. More directly, Szymanski et al. reported that immunization with glycoconjugates bearing the C. jejuni N-linked heptasaccharide led to a 10-log reduction in colonization of chickens, in association with the elicitation of glycandirected antibodies [85]. Thus, while considerable progress has been made, there is still much related to the roles played by protein glycosylation in Bacteria that remains to be elucidated.

Archaeal N-Glycosylation – Reasons to Sweeten at Extremes Like Bacteria, Archaea are also capable of both N- and O-glycosylation [6,86]. Yet, whereas Nglycosylation is currently restricted to delta/epsilon proteobacteria [5], it seems to be an almost universal process in Archaea [87]. Because of this, and since only three examples of archaeal O-glycosylation have been reported [88–90], the bulk of research into protein glycosylation in Archaea has focused on N-glycosylation. Such efforts have not only revealed novel aspects of this universal post-translational modification but have also provided insight into what purpose it serves in Archaea. Having provided the first example of a non-eukaryal glycoprotein [88], halophilic (salt-loving) archaea have long served as models of choice for understanding N-glycosylation in Archaea. As such, not only are halophiles central to research aimed at defining archaeal N-glycosylation Box 2. Glycoengineering with Bacteria: Escherichia coli [201_TD$IF]As a Glycofactory With the description of a pathway for N-glycosylation in Campylobacter jejuni [14], efforts subsequently focused on introducing the ability to glycosylate proteins into E. coli, a bacterial workhorse of biotechnology. Accordingly, E. coli transformed with the gene cluster encoding components of the C. jejuni N-glycosylation pathway was able to glycosylate recombinant C. jejuni glycoproteins, as well as proteins into which a sequence recognized by the Nglycosylation machinery was inserted [68,117]. It was also reported that similar N-glycosylation was performed by E. coli expressing the C. jejuni oligosaccharyltransferase PglB and enzymes involved in the assembly of different O-polysaccharides, components of the lipopolysaccharide surrounding Gram-negative bacteria, demonstrating that variability of the glycan added to target proteins in engineered E. coli was possible [118]. More recently, efforts have focused on generating E. coli strains capable of producing glycoproteins bearing human N-linked glycans. In such efforts, genes encoding yeast enzymes common to the yeast and human N-glycosylation pathways have been introduced into E. coli cells expressing C. jejuni PglB, yielding proteins bearing the core N-linked glycan of eukaryotic glycoproteins [119]. Although much remains to be done before E. coli can serve as host cell for economically viable glycoprotein production, considerable efforts are being directed at optimizing glycosylation efficiency and glycoprotein yield, as well as humanizing the added glycans.

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Parent

pathways [6], they have also helped elucidate the reasons for this post-translational modification. At present, these efforts have largely focused on three haloarchaeal glycoproteins, namely surface (S)-layer glycoproteins that comprises the S-layer surrounding such cells, archaellins, the building blocks of the archaellum (corresponding to the archaeal counterparts of bacteria flagellins and flagella, respectively), and pilins, the basic subunit of pili, another surface appendage found in haloarchaea [91–95]. Such studies have revealed that N-glycosylation is important for the integrity and strength of the structures assembled from these glycoproteins. In some of the earliest studies on archaeal N-glycosylation, it was shown that treating Halobacterium salinarum with bacitracin, a compound known to interfere with other glycosylation systems, not only affected S-layer glycoprotein mass but also converted these normally rod-like cells into spheres [96]. More recently, it was shown that interfering with proper Haloferax volcanii N-glycosylation through the deletion of genes encoding proteins involved in this post-translational event rendered the S-layer more susceptible to proteolytic degradation or more readily released to the surrounding medium, apparently due to under-glycosylation of the S-layer glycoprotein [97–102]. The importance of N-glycosylation for Hfx. volcanii S-layer integrity was further shown with membrane vesicles prepared from cells deleted of N-glycosylation pathway genes; such vesicles presented only partially intact S-layers [102]. Moreover, it was reported that truncation of the pentasaccharide N-linked to the Hfx. volcanii S-layer glycoprotein resulted in reduced secretion of a reporter protein to the growth medium, presumably due to effects on the assembly or architecture of the S-layer [102] (Figure 2).

S-layer Plasma membrane

N-glycosylaon mutants

Enhanced sensivity to added protease Increased release into the growth medium

Loss of integrity upon vesicle formaon

Decreased protein export to the medium

Figure 2. Compromised N-Glycosylation Has a Detrimental Effect on the Hfx. volcanii S-Layer. The Hfx. volcanii S-layer surrounding the cell comprises a single protein, the S-layer glycoprotein (schematically depicted in the top panel, Parent strain). In mutants where N-glycosylation is perturbed by deletion of genes involved in the process, the Slayer shows increased sensitivity to added protease (second panel; [98–102]), is more readily released into the growth medium (third panel; [97]), loses its integrity when right-side-out membrane vesicles are prepared (fourth panel; [102]), and interferes with the export of protein to the growth medium (bottom panel; [102]).

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Compromised N-glycosylation also led to defects in the assembly of archaella and pili in Hfx. volcanii, as well as losses of motility and adhesion, demonstrating the functional importance of proper archaellin and pillin N-glycosylation, respectively [95,103]. The importance of archaellin N-glycosylation for archaellum assembly and motility was likewise demonstrated in the methanogenic archaea Methanococcus voltae [104] and Methanococcus maripaludis [105]. In the thermoacidophile Sulfolobus acidocaldarius, compromised N-glycosylation did not affect archaellum assembly, structure or stability, yet did compromise motility [106,107]. Disrupted N-glycosylation also interfered with proper S. acidocaldarius S-layer structure [107]. Moreover, perturbed N-glycosylation substantially decreased S. acidocaldarius growth rates as the salinity of the surrounding medium increased [106,108]. It is noteworthy that [207_TD$IF]in S. acidocaldarius, a species belonging to the major archaeal phylum Crenarchaeota, Nglycosylation is essential for survival [109], whereas this is not the case in Euryarchaeota, a second major archaeal phylum that includes the halophiles and methanogens discussed above [104,110]. More striking than the contribution of N-glycosylation to the architecture, integrity, and function of glycoprotein-based structures is the apparent involvement of this protein-processing event in archaeal responses to changing environments. Although many Archaea are denizens of ‘extreme’ surroundings, they too can experience environment-related stress, as conditions in such habitats may not remain static. Post-translational modifications, including protein glycosylation, provide a rapid and reversible response to such environmental changes. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pentasaccharide [13]. However, when the salinity of the growth medium dropped below a certain threshold, both dolichol phosphate, the lipid upon which N-linked glycans are assembled in Hfx. volcanii, and S-layer glycoprotein Asn-498, a position not modified when the cells are grown at higher salinity, were modified by a tetrasaccharide of novel composition [111–113]. Moreover, if assembly of the pentasaccharide was compromised, the so-called ‘low salt’ tetrasaccharide was added to Asn-498 even under high-salt conditions, indicative of the coordinated behavior of the two Nglycosylation pathways in this organism. In other studies, it was reported that the N-glycosylation profile of M. maripaludis archaellins was modified when the growth temperature exceeded a certain threshold [114]. Whereas the archaellin FlaB is normally modified by a tetrasaccharide at its four N-glycosylation sites, in cells grown at temperatures [208_TD$IF]38 C, this protein was instead modified by only the first three sugars, with sugar three lacking the attached Thr residue and acetamidino group seen at this position at lower temperatures. It was further confirmed that the appearance of the truncated N-linked glycan was not the result of tetrasaccharide degradation at the elevated growth temperature. These results suggest that modulating the N-glycosylation pattern of proteins that undergo such post-translational modification somehow helps Archaea adapt to changes in the environment. It remains, however, to be determined just how modified N-glycosylation contributes to such adaptability. With this in mind, it is worth noting that in Hbt. salinarum, the transcription factor TrmB regulates the expression of metabolic enzymes, including those involved in the production of sugars used in protein glycosylation, in response to glucose levels [115]. In summary, novel uses for protein glycosylation by Archaea may reflect yet another creative solution these organisms employ to cope with the extreme environments in which they are often found.

Concluding Remarks Protein glycosylation is of undoubtable biological significance in a variety of prokaryotes. However, current studies into protein glycosylation clearly represent only the tip of an iceberg and can be considered as having addressed the ‘low hanging fruit'. As seen by mutant analyses in Bacteria, the phenotypes are all over the map and in a number of cases, lack coherence. In Archaea, the limited number of species amenable to experimental manipulation represents an

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Outstanding Questions What additional roles does protein glycosylation serve in prokaryotes? Do eukaryotes also rely on protein glycosylation for purposes now seemingly unique to prokaryotes? Why are there so many different bacterial protein glycosylation systems? Do the innate and/or adaptive immune systems provide the driving force or are metabolic considerations relating to the costs of synthesizing certain oligosaccharides responsible? How widespread is the use of modified N-glycosylation as a response to environmental change in Archaea? How does modified N-glycosylation contribute to the ability of Archaea to adapt to environmental changes? Do Bacteria also modify protein glycosylation in response to changing surroundings? Does O-glycosylation change in the face of shifting environments? How can prokaryotic protein glycosylation be exploited for applied purposes?

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obstacle to the drawing of general conclusions. As such, although evidence for new and seemingly prokaryote-specific roles for protein glycosylation is starting to accumulate, it remains a challenge to define precise functions for this post-translational modification in such organisms (see Outstanding Questions). This is not new. In 1993, Varki summarized the potential biological significance of oligosaccharides with the conclusion that “all of the theories are correct” [116]. When one includes prokaryotic protein glycosylation into the equation, this conclusion may be more true now than ever. Acknowledgments J.E. was supported by grants from the Israel Science Foundation (ISF) (grant 109/16), the ISF within the ISF-UGC joint research program framework (grant 2253/15), the ISF-NSFC joint research program (grant 2193/16) and the GermanIsraeli Foundation for Scientific Research and Development (grant I-1290-416.13/2015). [209_TD$IF]M.K. was supported in part by Research Council of Norway (RCN) project 214442, as well as the Centre for Integrative Microbial Evolution at the Department of Biosciences, University of Oslo.

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