Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti

Abstract

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395-and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar fiatnents. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N-and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.Bacterial flagella consist of a helical filament, a proximal hook, and a basal body with the flagellar motor (31). The long helical filaments are polymers assembled from flagellin subunits, whose molecular weights range between 20,000 and 65,000, depending on the bacterial species (5,9,23,25,27,34,35,38).Two types of flagellar filaments, named plain and complex, have been distinguished by their electron microscopically determined surface structures (42). Plain filaments have a smooth surface structure with faint helical lines, whereas complex filaments exhibit a conspicuous helical pattern of alternating ridges and grooves (13,41,42,46). These helical undulations are considered to be responsible for the brittle and (by implication) rigid structure (46) that enables them to propel bacteria efficiently in viscous media (13). This may reflect environmental selection, since complex flagella have been observed in only three species of soil bacteria: Pseudomonas rhodos (42), Rhizobium lupini H13-3 (35, 41), and R. meliloti (13,27). Plain filaments have been observed in most well-studied swimming bacteria, such as Escherichia coli, Salmonella typhimurium, and Bacillus subtilis. Whereas flagella with plain filaments can alternate between clockwise and counterclockwise rotation (31, 43), all known flagella with complex filaments rotate only clockwise with intermittent stops (14).Measurements of mass per unit length and three-dimensional reconstruction from electron micrographs suggested that the complex filaments of R. lupini are composed of functional dimers, whereas the plain filaments of S. typhimurium consist of functional monomers (46,47). This is consistent with the present finding in R. meliloti of two flagellin genes, named flaA and flaB, that encode closely related proteins. We propose that the complex filaments are composed of heterodimeric subunits that require stoichiometric amounts of the flaA and flaB gene products. Curiously, the two tandemly arranged genes were shown to be transcribed individually from unusual pr...

Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence

“…The sequencing of the latter protein revealed variations in the fifth and eighth amino acid residues, which suggests that two proteins may have co-migrated in the polyacrylamide gel. The sequences are very similar to the amino acid N-terminus sequence of flagellin encoded by flaA and flaB genes of Rhizobium meliloti (Pleier and Schmitt, 1989). These results therefore suggest that the major proteins released from A. tumefaciens cells in liquid culture represent flagellar proteins.…”

“…Flagellar proteins are frequently the major proteins that occur in culture supernatants because flagellated bacteria shed their flagella through mechanical agitation. Our analysis of the prominent proteins appearing in the culture supernatant proved them to be those from flagella because of the following: (i) the amino acid sequence of the N-terminus of these proteins showed homologies to the published FlaA and FlaB sequence of R. meliloti (Pleier and Schmitt, 1989); (ii) the nucleotide sequence of the isolated ORFs corresponding to flaA and flaB genes were similar (by an average of 66% identity) to those of R. meliloti; and (iii) the N-terminal amino acid sequence predicted from these ORFs matched exactly with the amino acid sequence analysis of the two isolated proteins. Sequence analysis of the cloned DNA bearing flaA and flaB also revealed a third ORF whose sequence was similar to flaA and flaB but contained dissimilar sequences in the central region of the ORF.…”

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella

“…In this property of differential spatial localization of flagellins in the filament, the flagella of H. pylori and Caulobacter species are unusual. For example, in the case of Rhizobium meliloti, it is proposed that the complex flagellar filaments are composed of heterodimeric subunits that require stoichiometric amounts of the two flagellins (43), while in other cases in which there are multiple flagellin species produced by an organism, their organization in the filament has not been determined.…”

Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

“…A putative σ 28 -dependent promoter was located immediately upstream of orf5 (data not shown). Therefore, V. cholerae may express multiple flagellin subunit proteins, as do many other motile Gram-negative bacterial species (Nuijten et al, 1990;Pleier and Schmitt, 1989).…”