The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant posttranslational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel Nlinked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of ␤-ManpNAcA6Thr-(1-4)-␤-GlcpNAc3NAcA-(1-3)-␤-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.Glycosylation of prokaryotic proteins is now well accepted, and examples of N-and O-glycosylation and of attachment of glycosylphosphatidylinositol anchors can now be found in the literature (1, 2). However, in contrast to eukaryotic glycosylation systems, prokaryotic systems display considerable diversity in the structure of the respective glycans and the proximal monosaccharide linkage. As a consequence there is considerable interest in determining the structural and genetic basis of glycan production among these diverse prokaryotic systems.The archaeal flagellum is a unique motility structure that is distinct from the well characterized bacterial flagellum (3). In contrast to bacterial flagellar assembly where newly synthesized flagellin is incorporated at the distal tip of the filament, it is believed that mature archaeal flagellin is incorporated at the base of the filament. In recent studies, the assembly of archaeal flagellum has been shown to more closely resemble a second bacterial motility system, the type IV pilus, where the structural protein pilin is synthesized with an unusual signal peptide and a hydrophobic N terminus. In Archaea, signal peptidases have been shown to cleave a signal peptide of the preflagellin proteins to produce mature flagellin, which is then incorporated into the filament (4). Flagellated archaeal species have one to five flagellin genes organized into a fla locus (3). The marine archaeon Methanococcus voltae has four flagellin structural genes organized in two transcriptional units: one unit contains flaA, whereas the second unit contains flaB1, flaB...

Prokaryotes use a wide variety of structures to facilitate motility. The majority of research to date has focused on swimming motility and the molecular architecture of the bacterial flagellum. While intriguing questions remain, especially concerning the specialized export system involved in flagellum assembly, for the most part the structural components and their location within the flagellum and function are now known. The same cannot be said of the other apparati including archaeal flagella, type IV pili, the junctional pore, ratchet structure and the contractile cytoskeleton used by a variety of organisms for motility. In these cases, many of the structural components have yet to be identified and the mechanism of action that results in motility is often still poorly understood. Research on the bacterial flagellum has greatly aided our understanding of not only motility but also protein secretion and genetic regulation systems. Continued study and understanding of all prokaryotic motility structures will provide a wealth of knowledge that is sure to extend beyond the bounds of prokaryotic movement. OverviewMotility is widespread throughout the prokaryotes, yet no one structure confers motility to all organisms in all circumstances. Of the motility structures, the bacterial flagellum has received the most attention from researchers. There exist a number of variations on the classical flagellum, such as different lateral and polar flagella on the same cell, and the periplasmic flagella of spirochaetes. Motility is also conferred by flagella in the domain Archaea, yet these structures bear little similarity to their bacterial counterparts. Rather, archaeal flagella demonstrate similarity to another bacterial motility apparatus, type IV pili. Additional structures involved in bacterial motility include the junctional pore complex and the ratchet structure involved in gliding motility, and the unique contractile cytoskeleton of Spiroplasma. Bacterial flagellaWithout a doubt the most common and best studied of all prokaryotic motility structures is the bacterial flagellum (Aldridge & Hughes, 2002;Macnab, 1999). Composed of over 20 protein species with approximately another 30 proteins required for regulation and assembly, it is one of the most complex of all prokaryotic organelles (Fig. 1). Well understood in its own right as a motility structure, it has become a model system for type III secretion systems in general (Aldridge & Hughes, 2002).The bacterial flagellum is a rotary structure driven from a motor at the base, with the filament acting as a propeller. The flagellum consists of three major substructures: the filament, the hook and the basal body. The filament is typically about 20 nm in diameter and usually consists of thousands of copies of a single protein called flagellin. Less commonly the filament is composed of several different flagellins. At the tip of the flagellum is the capping protein HAP2. Connecting the filament to the basal body is the hook region, composed of a single protein. The junction ...

SummaryThe 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.

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.

While they are functionally similar, archaeal flagella have characteristics not typically seen in bacterial flagella (37). Archaeal flagellins are synthesized with a leader peptide (3, 18), which is cleaved prior to the incorporation of the flagellin into the filament, similar to the processing of type IV pilins before incorporation into the pilus (34). A Methanococcus maripaludis protein (FlaK) possessing preflagellin peptidase activity has recently been reported (2). Archaeal flagellins show sequence similarity to type IV pilins at the N termini of the mature proteins (8) and do not demonstrate homology to bacterial flagellins. Archaeal flagella are thinner in diameter (10 to 13 nm [4,16,33]) than bacterial flagella (20 nm [17]) and are always composed of multiple flagellins, which are often glycosylated (6,20,24). Additionally, a search of completely sequenced archaeal genomes failed to identify genes homologous to any genes coding for structural proteins involved in bacterial flagellation (7). This includes, but is not limited to, genes encoding the hook, rod, or ring proteins. All of these characteristics suggest that the structural components of the archaeal flagella are composed of unique, archaeon-specific proteins, possibly fulfilling the same function as those present in bacterial flagella, and that the mode of assembly is likely distinct as well.Methanococcus voltae is a marine organism possessing more than 70 flagella on the cell surface. As is typical of archaeal flagella, M. voltae flagella are composed of multiple flagellins (18). There are four flagellin genes found within two transcriptional units in the M. voltae chromosome, with the first transcriptional unit containing a single flagellin gene, flaA. The second transcriptional unit includes the three remaining flagellin genes (flaB1, flaB2, and flaB3) and the downstream cotranscribed flagellar accessory genes flaCDEFGHIJ (18; N. A. Thomas and K. F. Jarrell, unpublished data). Purified flagella were shown to be composed of two major proteins, flagellins FlaB1 and FlaB2, with molecular masses corresponding to 33 and 31 kDa, respectively (18). Prior to the work presented in this study, the remaining two flagellins (FlaA and FlaB3) remained undetected.Within the flagellated archaea, little work has been done to address the universal presence of multiple flagellins. In Halobacterium salinarum, five flagellin genes are arranged in two different loci. Two flagellin genes (flgA1 and flgA2) are arranged in tandem at one locus, with the remaining three genes (flgB1, flgB2, and flgB3) clustered in another locus, and all five corresponding gene products have been identified within isolated flagella (9). The estimated lengths of the mRNAs indicate that the flagellin genes within each locus are cotranscribed, but the transcripts did not include the accessory genes as seen in M. voltae and other methanogens (10, 38). It was recently determined that the majority of the accessory genes observed in M. voltae are present in H. salinarum next to the flgB locus bu...