SummaryThe archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella-associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function (flaC, flaD, flaE, flaF, flaG and flaH), an ATPase (flaI ) and a membrane protein (flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full-length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella-associated genes. Markerless in-frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the DFlaB3 and FlaD M(191)I strains, all mutants were non-motile by light microscopy and non-flagellated by electron microscopy. A detailed examination of the DFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function.

Bradyrhizobium japonicum is one of the soil bacteria that form nodules on soybean roots. The cell has two sets of flagellar systems, one thick flagellum and a few thin flagella, uniquely growing at subpolar positions. The thick flagellum appears to be semicoiled in morphology, and the thin flagella were in a tight-curly form as observed by dark-field microscopy. Flagellin genes were identified from the amino acid sequence of each flagellin. Flagellar genes for the thick flagellum are scattered into several clusters on the genome, while those genes for the thin flagellum are compactly organized in one cluster. Both types of flagella are powered by proton-driven motors. The swimming propulsion is supplied mainly by the thick flagellum. B. japonicum flagellar systems resemble the polar-lateral flagellar systems of Vibrio species but differ in several aspects.Bradyrhizobium japonicum is a nitrogen-fixing bacterial species that forms root nodules specifically on soybean (Glycine max) roots. Because soybeans are a good dietary source of protein for vegetarians, soybeans and hence B. japonicum are agriculturally important. The complete genome of B. japonicum has been sequenced (8). It retains both flagellar and type III secretion systems, which play a crucial role in plant-microorganism interactions, especially for bacterial adhesion to the root hair surfaces.Flagella of rhizobacterial species are different from those of enteric species. For example, Rhizobium lupini or Sinorhizobium meliloti has peritrichous flagella, but the flagellar filament shows a zigzag pattern on the surface, which is called the complex filament. The complex filament exhibits a prominent helical pattern of alternating ridges and grooves, thus appearing more complex than plain filaments of enteric bacteria (14,16). Azospirillum brasilense has two sets of flagellar systems (6): a polar flagellum and lateral flagella, similar to those of Vibrio parahaemolyticus (4, 11). V. parahaemolyticus cells swim in aqueous (low-viscosity) conditions by using a single polar flagellum as a screw, while they swarm on the viscous surface by using lateral flagella (9).In this study, we first observed B. japonicum cells by electron microscopy and were amazed about the unusual set of flagella, one thick flagellum and a few thin flagella, both growing from the side of the cell body. Thus, they are distinctive from the similar set of flagella of V. parahaemolyticus: polar flagellum and lateral flagella. We have purified the flagella separately from mutants, analyzed the component proteins by amino acid sequencing, and identified the genes encoding those proteins. We have also examined the role of each flagellum by microscopic observations of mutants that carry only one set of the flagella. MATERIALS AND METHODSBacterial strains and growth conditions. B. japonicum strain 110spc4 is a mutant derivative of B. japonicum USDA3I1b110. BJD⌬283 is a mutant with the deletion of flagellin genes bll6865 and bll6866, which are part of the set 2 cluster of flagellar genes. They en...

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.

When motile swarmer cells of Caulobacter crescentus differentiate into sessile stalked cells, the flagellum is ejected. To elucidate the molecular mechanism of the flagellar ejection, flagellar hook-basal body (HBB) complexes from C. crescentus were purified and characterized. The purified HBBs were less stable against acidic pH or protease treatment than HBBs of Salmonella typhimurium, supporting the view that flagellar ejection from C. crescentus is initiated by destruction of the fragile basal structures. In addition, protease treatment of the purified flagella resulted in the specific digestion of the MS ring complex, revealing for the first time the intact structure of the whole rod.