Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

Abstract

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.

Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens

“…Comparison of the N-terminal sequence of one purified flagellin with the amino acid sequence predicted from the gene sequence (13) confirmed the presence of a 12-amino-acid leader peptide. Similarly, in the related methanogen Methanococcus vannielii, comparison of the N-terminal sequences obtained from the two major flagellins of purified flagellar filaments with the deduced amino acid sequence of the cloned genes indicated the presence of 12-amino-acid leader peptides on the FlaB1 and FlaB2 flagellins (2). The presence of leader peptides on archaeal flagellins indicated that enzymatic activity must be present in archaeal cells to process the preflagellins.…”

“…However, recent evidence has indicated that the archaeal flagellum is a unique motility structure, distinct from that of bacteria in composition and likely assembly (4,10). One major distinction is that the assembly of archaeal flagella requires the posttranslational cleavage of a short (11-or 12-amino-acid) leader peptide (2,13) from the precursor form of the flagellin monomer (preflagellin) before its incorporation into the growing filament. Bacterial flagellins are not made with leader peptides (12).…”

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

“…It has been demonstrated for some methanogens that leader peptides are cleaved from preflagellins upon export (2,14,24). This is likely true of all archaeal flagellins, based on sequence alignments (45).…”

“…Although the multiple flagellin genes within a particular family share considerable sequence similarity, mutant studies with Methanococcus voltae and H. salinarum suggest that the proteins encoded by the multiple flagellin genes are not simply interchangeable and likely have specific roles within the filament (23,44). This is supported by the fact that archaeal flagellar filaments tend to be composed of a mixture of different flagellins, present in varying amounts (2,16).…”

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

“…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).…”