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A molecular ruler, FliK, controls the length of the flagellar hook. FliK measures hook length and catalyses the secretion-substrate specificity switch from rod-hook substrate specificity to late substrate secretion, which includes the filament subunits. Here, we show normal hook-length control and filament assembly in the complete absence of the C-ring thus refuting the previous ‘cup’ model for hook-length control. Mutants of C-ring components, which are reported to produce short hooks, show a reduced rate of hook–basal body assembly thereby allowing for a premature secretion-substrate specificity switch. Unlike fliK null mutants, hook-length control in an autocleavage-defective mutant of flhB, the protein responsible for the switch to late substrate secretion, is completely abolished. FliK deletion variants that retain the ability to measure hook length are secreted thus demonstrating that FliK directly measures rod-hook length during the secretion process. Finally, we present a unifying model accounting for all published data on hook-length control in which FliK acts as a molecular ruler that takes measurements of rod-hook length while being intermittently secreted during the assembly process of the hook–basal body complex. [ABSTRACT FROM AUTHOR]

Recently, the flagellin proteins of Methanococcus maripaludis were found to harbour an N-linked tetrasaccharide composed of N-acetylgalactosamine, di-acetylated glucuronic acid, an acetylated and acetamidino-modified mannuronic acid linked to threonine, and a novel terminal sugar [( 5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L- erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in the assembly and attachment of this glycan, in-frame deletions were constructed in putative glycan assembly genes. Successful deletion of genes encoding three glycosyltransferases and an oligosaccharyltransferase (Stt3p homologue) resulted in flagellins of decreased molecular masses as evidenced by immunoblotting, indicating partial or completely absent glycan structures. Deletion of the oligosaccharyltransferase or the glycosyltransferase responsible for the transfer of the second sugar in the chain resulted in flagellins that were not assembled into flagella filaments, as evidenced by electron microscopy. Deletions of the glycosyltransferases responsible for the addition of the third and terminal sugars in the glycan were confirmed by mass spectrometry analysis of purified flagellins from these mutants. Although flagellated, these mutants had decreased motility as evidenced by semi-swarm plate analysis with the presence of each additional sugar improving movement capabilities. [ABSTRACT FROM AUTHOR]

The 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 ΔFlaB3 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 ΔFlaB3 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. [ABSTRACT FROM AUTHOR]

We analysed all major proteins secreted into culture media from Salmonella typhimurium . Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS–polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a Δmot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is a motility apparatus in which a long helical filament - the propeller - is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed. [ABSTRACT FROM AUTHOR]