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

Abstract

Background: Rhizobium 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.

Results: R. 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.

Conclusion: The 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.

Figure 1

Sequence alignment of the seven flagellin subunits of R. leguminosarum bv. viciae strain 3841. Asterisks represent conserved residues; colons represent conserved substitutions; dots represent semi-conserved substitutions. The tryptic peptides detected in the upper band for 3841wt flagellar preparations are highlighted. FlaA peptides are highlighted in yellow; FlaB peptides are highlighted in gray; FlaC peptides are highlighted in teal. The peptides unique for the flagellin subunit are underlined. The glycosylation signals are in boxes. The sequence coverage of FlaA, FlaB, and FlaC are 44%, 37%, and 31%, respectively.

Figure 2

Alignment of R. leguminosarum VF39SM flagellin amino acid sequences. Asterisks represent conserved residues; colons represent conserved substitutions; dots represent semi-conserved substitutions. The tryptic peptides detected in the flagellar samples by tandem mass spectrometry are highlighted. FlaA peptides are highlighted in yellow; FlaB peptides are highlighted in light gray; FlaC peptides are highlighted in dark gray; FlaG peptides are highlighted in teal; FlaE peptides are highlighted in moss green. The peptides unique for each flagellin are underlined. The glycosylation signals are in boxes. The sequence coverage of FlaA, FlaB, FlaC, FlaG, and FlaE are 46%, 43%, 29%, 28%, and 18%, respectively.

Figure 3

Electron micrographs of R. leguminosarum and S. meliloti 1021 flagellar filaments stained with 1% uranyl acetate. (a) VF39SM is peritrichously flagellated; (b) 3841 has a subpolar flagellum; (c) S. meliloti 1021 is peritrichously flagellated. The flagellar filaments of (d) VF39SM and (e) 3841 appear to have a smooth surface and lack the ridging pattern observed on the surface of the complex flagella formed by (f) S. meliloti 1021. Bars: 500 nm for a, b and c; 100 nm for d, e and f.

Figure 4

Electron micrographs of R. leguminosarum VF39SM fla mutants stained with uranyl acetate. Inset pictures show the flagellar filaments at higher magnification. (a) flaA^- (b) flaB^- (c) flaC^- (d) flaD^- (e) flaE^- (f) flaH^- (g) flaG^- (h) flaB/C/D^- (i) flaA/B/C/D^-. Bars: 500 nm for cells with flagella; 100 nm for inset pictures.

Figure 5

Electron micrographs of R. leguminosarum 3841 fla mutants stained with uranyl acetate. Inset pictures show the flagellar filaments at higher magnification. (a) flaA^- (b) flaB^- (c) flaE^- (d) flaH^- Bars: 500 nm for cells with flagella; 100 nm for inset pictures.

Figure 6

Glycoprotein staining of R. leguminosarum flagellin proteins. A. Pro-Q Emerald 300 stain. Lane 1-Molecular marker. Molecular masses (in kDa) are shown on the left of panel B; Lane 2-CandyCane glycoprotein molecular weight standard, 42kDa α1-Acid glycoprotein served as a positive control (shown in panel A) and a 29kDa-protein, carbonic anhydrase (shown in panel B) served as a negative control for glycosylation; Lane 3 - VF39SM; Lane 4 - 3841. B. Coomassie Brilliant Blue stain to demonstrate total proteins. Same sample arrangement as in panel A.