Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

Abstract

BackgroundRhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.ResultsR. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility.ConclusionThe results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.

O-antigen and Core Carbohydrate of Vibrio fischeri Lipopolysaccharide

“…The flaD mutant is thought to be a "pure" motility mutant, without a predicted LPS biogenesis defect. 3 The precise structural effect of the flaD mutation is not known, but it could result in fewer or shorter flagellin filaments, without altering flagellar rotation (36,66).…”

Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor

“…In Sinorhizobium meliloti, motility-impaired mutants that lack flagella have a reduced attachment to plastic surfaces (29). Rle has one or two subpolar flagella, which are responsible for swimming and swarming motility (11,30). We investigated whether the abundance of flagellar proteins is modulated by light in Rle by SDS/PAGE of purified flagellar proteins (Fig.…”

“…S3). FlgE, FlaA, and FlaB have been shown to be present in the culture supernatant and/or in the flagellar filaments of R. leguminosarum strain 3841 (30,32). The presence of flagella was evaluated by means of SEM.…”

Distinct Cell Surface Appendages Produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, Cultivar-Specific and Nonspecific Symbionts of Soybean

“…The mass spectral data indicated that the 39-and 37-kDa bands present in USDA191 flagella had homology to FlaA and FlaD of S. meliloti, respectively. In contrast, both the 36-and 34-kDa bands from USDA257 (42). In order to examine if there were any differences in the number and amount of Nops present in the purified pilus preparations from USDA191 and USDA257, we performed side-by-side comparisons of the protein compositions of the pilus preparations (Fig.…”