Identification and Localization of Flagellins FlaA and FlaB3 within Flagella ofMethanococcus voltae

Abstract

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and flaB3) and the downstream cotranscribed flagellar accessory genes flaCDEFGHIJ (18; N. A. Thomas and K. F. Jarrell, unpublished data).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...

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

“…Two peptides for FlaA (13%) were also observed, including one glycopeptide (see Supplemental Table 1). These results are consistent with the fact that FlaA is the least abundant of the four flagellin components of the flagellar filament (6). In addition, FlaA may be resistant to enzymatic digestion as a consequence of its location within the flagellar filament.…”

“…To improve the coverage of the less abundant proteins the same analyses were carried out on intact flagellin preparations that retained a partial anchoring structure. It has been demonstrated previously that the FlaB3 protein is enriched in these preparations (6). Improved coverage was obtained for FlaB1 (77%), FlaB2 (98%), and FlaB3 (71%).…”

“…Early work demonstrated that FlaB1 and FlaB2 are the major species of the extended filament, whereas FlaB3 is localized proximal to the cell surface and is a major component of the curved hook region. The minor component FlaA is believed to be distributed throughout the filament (6).…”

A way to identify archaellins in Halobacterium salinarum archaella by FLAG-tagging

“…Furthermore, the relative content of archaellin B2 in the H. salinarum archaella is much higher than that of the hook-forming archaellin B3 in Methanococcus voltae. In the latter case, B3 is present in an amount hardly detected by electrophoresis of native archaella [11], while the share of H. salinarum archaellin B2 in wildtype archaella is about 15% that is apparently in excess for hook formation. It is possible that the presence of archaellin B2 along the whole length of the filament can someway stabilize the archaella helical structure [8].…”

“…Most studies on the role of multiple archaellins used methods of inactivation and deletion of genes [3]. Jarrell and co-workers have identified and localized minor archaellins FlaA1 and FlaB3 in the Methanococcus voltae filament, using antibodies specific to the variable regions of archaellins, and showed that FlaA1 is distributed over the entire length of the filament, while FlaB3 forms a short curved hook proximal to the cell [11].…”