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The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-β-GlcNAc3NGaAN-4-β-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide β-GlcNAc3NAcAN-4-β-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [−2-β-Ribf-3-α-Rha2OMe-3-α-Rha − 2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1–3)-[α-GalNAcA3OMe-(1–2)-]-β-Man-(1–4)-[β-GalA3OMe4OAc6CMe-(1–4)-α-GalA-(1–2)-]-α-GalAN-(1–3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography–MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis. This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.

Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate - lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarA Mma , was identified in Methanococcus maripaludis, one of the model organisms used for archaellum studies. EarA Mma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii, Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarA Mma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarAMma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.

The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are regulated under different growth conditions. In this study, we identify MMP1718 as the first transcriptional activator that directly regulates the fla operon in M. maripaludis. Mutants carrying an in-frame deletion in mmp1718 did not express FlaB2 detected by western blotting. Quantitative reverse transcription PCR analysis of purified RNA from the Δmmp1718 mutant showed that transcription of flaB2 was negligible compared to wildtype cells. In addition, no archaella were observed on the cell surface of the Δmmp1718 mutant. FlaB2 expression and archaellation were restored when the Δmmp1718 mutant was complemented with mmp1718 in trans. Electrophoretic motility shift assay and isothermal titration calorimetry results demonstrated the specific binding of purified MMP1718 to DNA fragments upstream of the fla promoter. Four 6 bp consensus sequences were found immediately upstream of the fla promoter and are considered the putative MMP1718-binding sites. Herein, we designate MMP1718 as EarA, the first euryarchaeal archaellum regulator.

The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.

Many archaea swim by means of archaella. While the archaellum is similar in function to its bacterial counterpart, its structure, composition, and evolution are fundamentally different. Archaella are related to archaeal and bacterial type IV pili. Despite recent advances, our understanding of molecular processes governing archaellum assembly and stability is still incomplete. Here, we determine the structures of Methanococcus archaella by X‐ray crystallography and cryo‐EM. The crystal structure of Methanocaldococcus jannaschii FlaB1 is the first and only crystal structure of any archaellin to date at a resolution of 1.5 A, which is put into biological context by a cryo‐EM reconstruction from Methanococcus maripaludis archaella at 4 A resolution created with helical single‐particle analysis. Our results indicate that the archaellum is predominantly composed of FlaB1. We identify N‐linked glycosylation by cryo‐EM and mass spectrometry. The crystal structure reveals a highly conserved metal‐binding site, which is validated by mass spectrometry and electron energy‐loss spectroscopy. We show in vitro that the metal‐binding site, which appears to be a widespread property of archaellin, is required for filament integrity.

Cell motility in pure cultures of halophilic archaea was first observed almost a century ago and, in methanogens, at least as long ago as 1951, when Methanococcus vannielii was first isolated. Although M. vannielii was reported as being motile, no flagella were observed until nearly three decades later. Other researchers from that era reported seeing fimbriae (pili) or flagella on a variety of different archaea, including methanogens, halophiles, and Sulfolobus. However, during that period the members of the archaea were still considered bacteria, predating the groundbreaking realignment of the tree of life into the three domains of Archaea, Bacteria, and Eukarya by Carl Woese.

Each of the three domains of life exhibits a unique motility structure: while Bacteria use flagella, Eukarya employ cilia, and Archaea swim using archaella. Since the new name for the archaeal motility structure was proposed, in 2012, a significant amount of new data on the regulation of transcription of archaella operons, the structure and function of archaellum subunits, their interactions, and cryo-EM data on in situ archaellum complexes in whole cells have been obtained. These data support the notion that the archaellum is evolutionary and structurally unrelated to the flagellum, but instead is related to archaeal and bacterial type IV pili and emphasize that it is a motility structure unique to the Archaea.

Methanococcus maripaludis is a stringently anaerobic archaeon with two studied surface structures, archaella and type IV pili. Previously, it was shown that three pilin genes (mmp0233 [epdA], mmp0236 [epdB] and mmp0237 [epdC]) located within an 11 gene cluster in the genome were necessary for normal piliation. This study focused on analysis of the remaining genes to determine their potential involvement in piliation. Reverse transcriptase PCR experiments demonstrated the 11 genes formed a single transcriptional unit. Deletions were made in all the non-pilin genes except mmp0231. Electron microscopy revealed that all the genes in the locus except mmp0235 and mmp0238 were essential for piliation. Complementation with a plasmid-borne wild-type copy of the deleted gene restored at least some piliation. We identified genes for an assembly ATPase and two versions of the conserved pilin platform forming protein necessary for pili assembly at a separate genetic locus.

The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are regulated under different growth conditions. In this study, we identify MMP1718 as the first transcriptional activator that directly regulates the fla operon in M. maripaludis. Mutants carrying an in-frame deletion in mmp1718 did not express FlaB2 detected by western blotting. Quantitative reverse transcription PCR analysis of purified RNA from the Δmmp1718 mutant showed that transcription of flaB2 was negligible compared to wildtype cells. In addition, no archaella were observed on the cell surface of the Δmmp1718 mutant. FlaB2 expression and archaellation were restored when the Δmmp1718 mutant was complemented with mmp1718 in trans. Electrophoretic motility shift assay and isothermal titration calorimetry results demonstrated the specific binding of purified MMP1718 to DNA fragments upstream of the fla promoter. Four 6 bp consensus sequences were found immediately upstream of the fla promoter and are considered the putative MMP1718-binding sites. Herein, we designate MMP1718 as EarA, the first euryarchaeal archaellum regulator.

Motility structures, called flagella, have been described in all three domains of life: Bacteria, Archaea and Eukarya. These structures are well studied in both Bacteria and Eukarya. However, already in eukaryotes there exists some confusion as to whether these structures should actually be called cilia. With increased studies conducted on organisms of the third domain of life, the Archaea, it has become clear that the archaeal flagellum only functionally appears similar to the bacterial flagellum, whereas it structurally resembles a bacterial type IV pilus. To resolve confusion due to unclear nomenclature, we propose renaming the archaeal flagellum as the 'archaellum'. This will make clear that the archaellum and the bacterial flagellum are two distinct structures that happen to both be used to enable microorganisms to swim.

Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific role has been assigned as yet to pili. In this report, wild-type M. maripaludis cells are compared with mutants lacking either pili or flagella or both surface appendages in their ability to attach to a variety of surfaces including nickel, gold and molybdenum grids as well as glass, silicon and mica. Wild-type cells attached to varying degrees to all surfaces tested, except mica, via their flagella as observed by scanning electron microscopy. Large cables of flagella were found to leave the cell and to be unwound on the surface. In addition, such cables were often found to connect cells. In contrast, cells lacking either flagella or pili or both surface appendages were unable to attach efficiently to any surfaces. This indicates a second role for flagella in addition to swimming in M. maripaludis, as well as a first role for pili in this organism, namely in surface attachment.

Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms,Haloferax volcanii, Methanococcus voltaeandMethanococcus maripaludis,has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for eitherHfx. volcaniior theMethanococcusspecies, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.

In Archaea , the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis ) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus , where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis Δ flaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis , a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal alpha-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by approximately 44 A and the other has a one-start helical symmetry with approximately 2.6 subunits per turn of a approximately 30 A pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.

Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.

The Mv1751 gene product is thought to catalyze the first step in the N-glycosylation pathway in Methanococcus voltae . Here, we show that a conditional lethal mutation in the alg7 gene ( N -acetylglucosamine-1-phosphate transferase) in Saccharomyces cerevisiae was successfully complemented with Mv1751, highlighting a rare case of cross-domain complementation.

In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities, and at a variety of growth temperatures. Of the conditions tested, western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. While the amount of FlaB2 was similar for cells grown below 35°C, protein levels decreased at 38°C and were barely detectable at 42°C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42°C. Electron microscopy confirmed that the FlaB2 levels detected by western blots corresponded to the state of archaellation, with cells grown at 42°C being mostly nonarchaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in western blots of cells grown at temperatures beyond 38°C suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. Mass spectrometry analysis of archaella isolated from cells grown at 40°C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the wildtype glycan.

In Methanococcus maripaludis, the three archaellins which comprise the archaellum are modified at multiple sites with an N-linked tetrasaccharide with the structure of Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-l-erythro-hexos-5-ulo-1,5-pyranose, so far found exclusively in this species. In this study, a six-gene cluster mmp1089–1094, neighboring one of the genomic regions already known to contain genes involved with the archaellin N-glycosylation pathway, was examined for its potential involvement in the archaellin N-glycosylation or sugar biosynthesis pathway. The co-transcription of these six genes was demonstrated by RT-PCR. Mutants carrying an in-frame deletion in mmp1090, mmp1091 or mmp1092 were successfully generated. The Δmmp1090 deletion mutant was archaellated when examined by electron microscopy and mass spectrometry analysis of purified archaella showed that the archaellins were modified with a truncated N-glycan in which the terminal sugar residue and the threonine linked to the third sugar residue were missing. Both gene annotation and bioinformatic analyses indicate that MMP1090 is a UDP-glucose 4-epimerase, suggesting that the unique terminal sugar of the archaellin N-glycan might be synthesised from UDP-glucose or UDP-N-acetylglucosamine with an essential early step in synthesis catalysed by MMP1090. In contrast, no detectable phenotype related to archaellin glycosylation was observed in mutants deleted for either mmp1091 or mmp1092 while attempts to delete mmp1089, mmp1093 and mmp1094 were unsuccessful. Based on its demonstrated involvement in the archaellin N-glycosylation pathway, we designated mmp1090 as aglW.

Methanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

The archaeal flagellum is a unique motility organelle. While superficially similar to the bacterial flagellum, several similarities have been reported between the archaeal flagellum and the bacterial type IV pilus system. These include the multiflagellin nature of the flagellar filament, N-terminal sequence similarities between archaeal flagellins and bacterial type IV pilins, as well as the presence of homologous proteins in the two systems. Recent advances in archaeal flagella research add to the growing list of similarities. First, the preflagellin peptidase that is responsible for processing the N-terminal signal peptide in preflagellins has been identified. The preflagellin peptidase is a membrane-bound enzyme topologically similar to its counterpart in the type IV pilus system (prepilin peptidase); the two enzymes are demonstrated to utilize the same catalytic mechanism. Second, it has been suggested that the archaeal flagellum and the bacterial type IV pilus share a similar mode of assembly. While bacterial flagellins and type IV pilins can be modified with O-linked glycans, N-linked glycans have recently been reported on archaeal flagellins. This mode of glycosylation, as well as the observation that the archaeal flagellum lacks a central channel, are both consistent with the proposed assembly model. On the other hand, the failure to identify other genes involved in archaeal flagellation by homology searches likely implies a novel aspect of the archaeal flagellar system. These interesting features remain to be deciphered through continued research. Such knowledge would be invaluable to motility and protein export studies in the Archaea.

The transport of proteins into and across hydrophobic membranes is an essential cellular process. The majority of proteins that are translocated in an unfolded conformation traverse the membrane by way of the universally conserved Sec pathway, whereas the twin arginine translocation pathway is responsible for the transport of folded proteins across the membrane. Structural, biochemical and genetic analyses of these processes in Archaea have revealed unique archaeal features, and have also provided a better understanding of these pathways in organisms of all domains. Further study of these pathways in Archaea might elucidate fundamental principles involved in each type of transport and could help determine their relative costs and benefits as well as evolutionary adaptations in protein secretion strategies.

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.

Summary The fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar-related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting data revealed that flaHIJ are co-transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non-polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non-motile and non-flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino-terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla− phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each ofMethanococcus voltae,Methanococcus maripaludis,Methanococcus thermolithotrophicus, andMethanococcus jannaschiihas been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes,flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes ofM. voltaewere successfully overexpressed as His-tagged proteins inEscherichia coli.These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionatedM. voltaecells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly,flaDwas found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes ofM. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.

Methanococcus voltaeis a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits.Escherichia colimembranes containing the expressedM. voltaepreflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species.M. voltaepreflagellin peptidase activity was optimal at 37°C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.

The highly conserved nature of the 5'-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (Mr = 30,800 and 28,600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1. is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 x 10(7) daltons. BCJA1 was stable over the pH range of 6-11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36500 and 28000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated.

Six strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from 2.7 km below the earth's surface in the Taylorsville Triassic Basin in Virginia. The cells of these strains were motile rods that were 1 to 1.1 μ in diameter and 2 to 5 μ long. The cells grew by oxidizing H2, formate, methanol (weakly), lactate (incompletely, to acetate and CO2), or pyruvate (incompletely) while reducing sulfate to sulfide; acetate did not serve as a catabolic substrate. Thiosulfate or sulfite could replace sulfate as an electron acceptor. The results of a phylogenetic analysis of the 16S rRNA gene indicated that these strains belong to the genus Desulfotomaculum, but are distinct from previously described Desulfotomaculum species. Thus, we propose a new species, Desulfotomaculum putei, for them, with strain TH-11 (= SMCC W459) as the type strain. The results of our phylogenetic analysis also indicated that strain SLTT, which was isolated from a hot spring and has been described previously (T. M. Karnauchow, S. F. Koval, and K. F. Jarrell, Syst. Appl. Microbiol. 15:296-310, 1992), is also a member of the genus Desulfotomaculum and is distinct from other species in this genus. We therefore propose the new species Desulfotomaculum luciae for this organism; strain SLT (= SMCC W644) is the type strain of D. luciae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.

N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5 S )-2-acetamido-2,4-dideoxy-5- O -methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes— mmp1084 , mmp1085 , mmp1086 , and mmp1087 —were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δ mmp1086 Δ mmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δ mmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δ mmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087 , either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV .

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.

Summary Three novel alkaliphilic bacteria were isolated and characterized following enrichment at pH 10, using mud from a bauxite-processing red mud tailing pond as source material. The isolates, designated strains JaA, JaD and JaH were aerobic, Gram-positive, spore-forming, mesophilic rods which were motile by means of peritrichously arranged flagella. Strains JaD and JaH could also grow anaerobically. All three isolates had pH optima for growth of approximately 10, with strains JaD and JaH obligate allcaliphiles. Strains JaA and JaD grew in the presence of 10% (w/v) NaCl while strain JaH could grow in the presence of up to 7.5% (w/v) NaCl. The mole %G+C content of purified DNA was 41.3% for strain JaA, 41.8% for strain JaD and 38.3% for strain JaH. The new isolates appear to be members of the genus Bacillus but 16s rDNA sequence analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparisons of whole cell protein profiles, as well as nutritional, biochemical and morphological traits indicated the new isolates are not members of any validly published Bacillus species. The epithet Bacillus vedderi is proposed for strain JaH.