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...

The complex flageilar filaments of Rhizobium meliloti are composed of two related (87% identical) flagellins that are encoded by closely linked, separately transcribed genes, flaA and flaB (E. Pleier and R. Schmitt, J. Bacteriol. 171:1467-1475. To elucidate the role of the subunits, A and B, in assembling the complex filament, the wild-type alleles were replaced with defective ones containing a 2,249-bp deletion (accompanied by substitution of a kanamycin resistance cartridge), which eliminates 74% offlaA (3' end) and 85% offlaB (5' end). The resulting nonmotile, filamentless mutant, RU110l1, was tested for complementation with wild-type flaA, flaB, and flaA flaB genes provided on the multiple-copy vector pRK290. Whereas flaA alone did not restore motility and filament production, both flaB and flaA flaB restored 20 to 30% of wild-type motility.Apparent causes of this reduced motility were fewer flagella per cell and/or shortened filaments sometimes ending in unusually thin, fragile structures. Tests with enzyme-linked antiflagellin antibodies indicated that faA is expressed at higher levels thanflaB and that multiple copies ofJfaA lead to reduced flagellin export. We conclude that the proximal portion of the complex filament is assembled from B subunits (not produced sufficiently to form full-length flagella) and that the distal portion is made from A subunits. Multiple copies of the strongflaA promoter may offset transcriptional controls that regulate the synthesis of flagellar structures required for flagellin export. The soil bacterium R. meliloti assembles between 5 and 10 flagella per cell (10). The filaments have an unusual complex fine structure exhibiting a conspicuous helical pattern of alternating ridges and grooves (10,29,30,36). The helical undulations are considered responsible for the rigid structure of complex filaments that enable them to propel bacteria efficiently in viscous media (10). On the basis of mass-perunit-length measurements and three-dimensional reconstruction from electron micrographs, it has been suggested (36) that the closely related complex filaments of Rhizobium lupini (10, 24) are composed of functional dimers, whereas the plain filaments of S. typhimurium consist of functional monomers (37).In resembled the so-called a8 promoters of Bacillus subtilis (9, 28).In an effort to better understand the process of assembling the complex filament of R. meliloti from two subunits, a genetic approach was chosen. Complementation analyses in a flagellin-deficient mutant strain with either flaA or flaB demonstrated that B subunits can form the proximal portion of the complex filament (and, conceivably, A subunits can form the distal portion). TheflaA gene is expressed at higher levels than flaB, and multiple copies of flaA in a cell obviously offset transcriptional controls, ultimately resulting in the down regulation of flagellin export. MATERIALS AND METHODSBacterial strains and plasmids. E. coli K-12 and R. meliloti MVII-1 derivatives (19) and the plasmids used are listed in Tables 1...

The complex flagella of Rhizobium meliloti 2011 and MVII-1 were analyzed with regard to serology, fine structure, subunits, and amino acid composition. The serological identities of flagellar filaments of the two strains were demonstrated by double immunodiffusion with antiflagellin antiserum. The filaments had a diameter of 16 nm. Their morphology was dominated by the prominent undulations of an external three-start helix running at a 10-nm axial distance and at an angle of 320. Faint nearly axial striations indicated the presence of a tubular core of a different helical order. The complex filaments consisted of 40,000-dalton flagellin monomers. Typically, the amino acid composition was 3 to 4% higher in nonpolar residues and 5 to 7% lower in aspartic and glutamic acids (and their amides) than that of plain flagellar proteins. There were no immunochemical relationships among Pseudomonas rhodos, Rhizobium lupini, and R. meliloti complex flagella, suggesting that the latter represent a new class.