Mass Spectrometry Unmasks Mystery Methanococcus Pilin

In this issue, Ng and colleagues describe their outside-in identification of genes involved in the expression of unusual type IV pili in the archaeon Methanococcus maripaludis (13). This intriguing tale, like many in science, hints at chapters not yet written and contains a common theme…expected the unexpected.

Prokaryotes, both bacteria and archaea, express a variety of pili, fimbriae, flagella, and even more exotic surface structures (11) used for adherence, cell-cell interactions, horizontal gene transfer, and motility. With the advent of rapid whole-genome sequencing, it has become possible to pick out the genes that could potentially encode the components of such structures by homology searches and to appreciate the extent of their distribution and conservation among genetically distant lineages (12).

In bacteria, type IV pili (T4P) are one of the most widely distributed classes of pilus (15). They were reported in Gram-negative genera such as Pseudomonas and Vibrio in the 1950s (14) and more recently in Gram-positive genera such as Clostridium (22). They can be either chromosomally or plasmid encoded and can be carried by horizontally transferable elements such as genetic islands (3, 8), potentially explaining their broad phylogenetic distribution. T4P are composed of pilin subunits that share a conserved hydrophobic N terminus containing a type III signal sequence that is recognized and cleaved on the cytoplasmic side of the plasma membrane by dedicated prepilin aspartyl peptidases (17). The highly conserved type III signal sequence (type I signal sequences are those recognized by signal peptidase I, while type II signal sequences are associated with lipoproteins) is a defining feature of the type IV pilin family.

Early investigations of archaeal surface organelles involved in swimming motility led to the surprising discovery that they are architecturally related to T4P, rather than to bacterial flagella (6). Archaeal flagella are composed of one or more subunit types that undergo the same kind of signal sequence processing as type IV pilins, by dedicated preflagellin peptidases (PibD in Sulfolobus and Haloferax and FlaK in Methanococcus) (7, 18, 20). In an archaeon-specific twist, the flagellin subunits must be both processed and modified by N-glycosylation prior to their assembly into a fiber (4). Although bacterial type IV pilins have been reported to undergo various posttranslational modifications, including glycosylation, those identified to date have been O-linked moieties (9). The similarities between archaeal flagellins and bacterial type IV pilins, coupled with electron microscopy data suggesting that archaeal flagella are unlikely to be hollow (in contrast to bacterial flagella), suggested that flagellar assembly in archaea might proceed by a T4P-like mechanism where new subunits are added to the base, not the tip, of the growing fiber (1).

Although decades of electron microscopy evidence showed that archaea had both thicker flagella and thinner pilus-like structures (also called fimbriae) on their surfaces, the specific functions of the latter structures and the details of their composition and biosynthesis remained enigmatic until very recently, when they were implicated in adhesion (19). Bioinformatic analyses of available archaeal genomes gave tantalizing clues to their biosynthetic origins, as genes encoding multiple proteins with putative type III signal sequences, some of which were experimentally verified to undergo processing, could be identified (18). For Ng and colleagues, it was advances in genetic techniques to allow in-frame deletion of flagellum biosynthesis genes in M. maripaludis that paved the way to the characterization of its thinner, less abundant pili.

Earlier structural studies of M. maripaludis pili by Wang et al. (24) revealed fibers with two distinct architectures, both of which could be visualized in the same filaments. It was not clear whether the filament forms represented two conformational states generated by different interactions among the same subunits (as has been observed for bacterial flagella) (16) or two distinct fiber forms resulting from the asymmetrical distribution of multiple subunits, a possibility based on the identification of multiple genes encoding pilin-like proteins with type III secretion signals in the M. maripaludis genome (5). Earlier this year, Biais and colleagues (2) showed that bacterial T4P can undergo force-dependent stretching deformations resulting in localized regions in which the fibers are elongated and narrower and new epitopes are exposed. It is possible that a similar phenomenon could explain the variation in pilus structures observed in the M. maripaludis pili. The identification of a possible lumen within M. maripaludis pili, regardless of their conformational state, was another interesting structural finding (24), as it contrasted with the dense core packing observed for bacterial T4P and archaeal flagella. Whether a fiber is hollow has implications for the mode of its assembly (from the base or at the tip via subunits secreted through the lumen) and potentially its function(s).

By deleting the M. maripaludis flaK gene to block flagellar assembly, Ng and coworkers were able to purify pili for biochemical analysis in order to match specific pilus subunits with the pilin-like genes identified by bioinformatic analyses of the M. maripaludis genome. A cluster of genes encoding three pilin-like proteins (MMP0233, MMP0236, and MMP0237) and the prepilin peptidase homologue EppA was a logical starting point. MMP0236 and MMP0237 appeared to have structural roles, as the mutants were bald until complemented. In contrast, MMP0233 mutants had fewer pili, while complemented mutants often had levels of pili greater than that of the parent strain, suggesting a possible role in control of assembly. Standard biochemical attempts to connect the genes with their corresponding products, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), periodic acid-Schiff (PAS) staining for glycosylation and peptide sequencing, proved unsatisfying, as the observed major (17-kDa) and minor (15-kDa) pilus components migrated diffusely, stained poorly with Coomassie, and were N-terminally blocked for protein sequencing with what turned out to be a cyclized pyroglutamic acid.

With these issues, the investigators turned to mass spectrometry (MS) to clarify the identity of the major pilin subunit. After some patient and creative modification of the methodology to digest and solubilize the pilus subunits, the investigators were able to obtain a mass of ∼9.7 kDa for the major subunit, though the complexity of the spectra hinted at the presence of extensive posttranslational modifications. Although glycosylation of the subunits was considered at the outset, based on the unusual migration and staining patterns in SDS-PAGE, standard PAS staining was negative.

Further tandem MS (MS/MS) studies revealed two unexpected results: the major subunit was none of the three previously considered proteins but instead a completely different pilin-like protein encoded by the unlinked MMP1685 locus, and its predicted mass, postprocessing, was only 6.4 kDa. With the sequence in hand, the investigators realized that the protein had only 1 Lys and no Arg, explaining their inability to generate useful tryptic fragments. The requirement of this protein for M. maripaludis piliation was confirmed by gene deletion, which led to bald mutants. Additional nano-liquid chromatography-MS/MS analysis showed that MMP1685 was modified with N-linked glycans very similar in composition to those previously identified on the flagellins of this species but with an additional branched hexose. This modification is the first example of an N-linked glycan reported for any pilin of the T4P family. Extensive glycosylation is the likely source of the difference between the predicted (6.4 kDa) and detected (9.7 kDa) masses of the major subunit and likely the reason for its aberrant migration and poor staining in SDS-PAGE experiments.

The use of MS approaches in this study was essential to pinpoint the identity of the major subunit of these novel pili, with the moral of the story being that genetic studies cannot reveal the entire picture. As with bacterial T4P, mutations in many of the genes involved in pilus assembly can lead to an identical nonpiliated phenotype, making it a challenge to ascribe functions to specific proteins. Even with SDS-PAGE information in hand, making straightforward predictions about which opening reading frame encodes the major pilin was not possible due to the unusually large apparent masses observed (∼17 kDa for the major species and ∼15 kDa for a minor species). Nor was it possible to confirm without MS data that the pilin subunit was glycosylated, since sugar-specific staining was negative. The surprisingly small size (only 62 residues) of MMP1685 and its lack of the conserved disulfide-forming Cys residues that are found in most type IV pilins are reminiscent of the Flp subclass of T4P (10), though MMP1685 has atypical features and may represent a new type of pilin.

Chapters in this story yet to be written include the identification of other components involved in the assembly of these novel pili; will they resemble those of previously characterized systems such as Flp, or will there be elements that are unique to archaea? The genes involved in N-glycosylation of the pilin remain to be characterized, as does the scope of their activities…will they turn out to be pilus specific or to have a broader role, as recently demonstrated for the Neisseria pilus glycosylation machinery (23)? One tip comes from a previous study of M. maripaludis in which mutants lacking an acetyltransferase involved in biosynthesis of the flagellin N-linked glycan had defects in pilus biogenesis; while pili were made, they were easily sloughed from the cells, suggesting that their assembly or anchorage was abnormal (21). This result, coupled with the similarity in glycan composition observed for the two systems, suggests that the archaeal flagellin and pilin posttranslational modification systems may overlap.

Archaea appear to have been early and enthusiastic adopters of the T4P paradigm. Close examination of the Sulfolobus solfataricus genome revealed, in addition to previously identified flagellin genes, additional open reading frames later shown to encode UV-inducible T4P (7), as well as a number of sugar-binding proteins with type III signal sequences (25). Additional pilin-like proteins of unknown function could be components of novel systems yet to be characterized. As genomic data from an array of diverse environmental habitats accumulate, it will be exciting to learn of even more unusual functions for which the versatile T4P platform has been adopted.