Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins
© Tambalo et al; licensee BioMed Central Ltd. 2010
Received: 10 April 2010
Accepted: 17 August 2010
Published: 17 August 2010
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.
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.
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.
Motility is an important property of bacteria that enables them to move towards favorable growth conditions and away from detrimental conditions. Most bacteria move through the use of flagella. A bacterial flagellum consists of three distinct regions: the basal body, the hook, and the filament . Flagellar assembly and motility are well-understood in enteric bacteria, particularly Escherichia coli and Salmonella. The flagellar filament of E. coli is a helical arrangement of as many as 20,000 flagellin subunits, whose molecular weight is approximately 50 kDa [1, 2]. Whereas the E. coli flagellar filament consists of one type of flagellin [3, 4], the presence of more than one flagellin type has been reported for a few soil bacteria, including Sinorhizobium meliloti, Rhizobium lupini, and Agrobacterium tumefaciens[5–10]. S. meliloti and A. tumefaciens assemble their flagellar filaments from four closely related flagellin subunits (FlaA, FlaB, FlaC, and FlaD) while R. lupini flagella consist of three flagellin subunits (FlaA, FlaB, and FlaD). For these soil bacteria, FlaA is the principal flagellin subunit of the flagellar filament while the other subunits play minor roles.
The flagellar filament is a highly conserved structure in terms of amino acid composition, subunit domain organization of the flagellin monomers, and the symmetry and mode of assembly [11, 12]. The quaternary structure of the flagellar filament has been divided into four structural domains, domain 0 (D0) to domain 3 (D3), and the amino acid residues of the flagellin protein have been assigned to these domains [13–17]. Domains D0 and D1, which are found in the filament core, correspond to the amino and carboxy terminal residues. Domains D2 and D3, the outer region of the filament, consist of the flagellin central residues. The amino acid sequences corresponding to domains 0 and 1 are highly conserved across different bacterial strains [14, 18], and were shown to be essential in the polymerization of bacterial flagellar filaments . Domains D2 and D3, on the other hand are considerably variable in amino acid composition and are generally not well-aligned . Domain D3 of the filament contributes to filament stability  but it can be deleted or reduced in size without severely impairing filament assembly and function [16, 20–22].
Flagellar filaments are traditionally classified as either "plain" or "complex". Plain filaments are often found in enterobacteria, such as Salmonella typhimurium and E. coli[23, 24]. These filaments have a smooth surface and are able to change from left- to right-handedness or from a counterclockwise to a clockwise direction of rotation . A few soil bacteria such as Pseudomonas rhodos, R. lupini[24, 26] and S. meliloti are equipped with one or more complex flagella. Studies have shown that transmission electron microscopy can be used to differentiate between plain and complex flagella [24, 27]. Complex flagellar filaments have a distinct ridging pattern while plain filaments appear thinner and have little to no visible external pattern. The complex filaments are also more rigid and more brittle than the plain filament. It is thought that increased rigidity is favorable for motility in viscous environment such as in the soil biotope .
To date, little is known about the flagellar filament of Rhizobium leguminosarum bv. viciae. A previous study has shown that the movement of R. leguminosarum bv. viciae strain 3841 is propelled by one or two subpolar flagella . The same study has also suggested that the flagella rotate in a unidirectional pattern and the direction of movement is changed by modulating the rotary speed. In this paper, we characterize the genes encoding the seven flagellin subunits in R. leguminosarum bv. viciae. We have conducted sequence analysis, as well as mutational and transcriptional studies to determine the roles of the flagellin genes in flagellar assembly and function for the sequenced strain 3841 and our laboratory strain VF39SM. We have studied the flagellin genes in parallel in both strains because the two strains exhibit differences in pattern of flagellation (see below) and also in swarming motility (below and ).
Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in the study.
Strains and Plasmids
Source or Reference
Escherichia coli strains
endA1, hsdR17, supE44, thi-1, recA1, gyrA96, relA1,(argF-lacZYA), U169, φ 80dlacZ ΔM15
Spr. RP4 tra region, mobilizer strain
Rhizobium leguminosarum strains
biovar viciae, JB300 derivative, Sm r
biovar viciae, Sm r
VF39SM flaA-, Sm r ,Nm r
VF39SMflaA- complemented with flaA, Sm r , Nm r ,Gm r
gusA-Nm cassette insertion in 3841 flaA, Sm r , Nm r
3841flaA- complemented with flaA, Sm r , Nm r ,Gm r
Spectinomycin cassette insertion in VF39SM flaB, Sm r ,Sp r
Spectinomycin cassette insertion in 3841 flaB, Sm r , Sp r
gusA-Nm cassette insertion in VF39SM flaC, Sm r , Nm r
gusA-Nm cassette insertion in 3841 flaC, Sm r , Nm r
gusA-Nm cassette insertion in VF39SM flaD, Sm r , Nm r
gusA-Nm cassette insertion in 3841 flaD, Sm r , Nm r
gusA-Nm cassette insertion in VF39SM flaE, Sm r , Nm r
gusA-Nm cassette insertion in 3841 flaE, Sm r , Nm r
Neomycin-resistance cassette insertion in VF39SM flaH, Sm r , Nm r
Neomycin-resistance cassette insertion in 3841 flaH, Sm r , Nm r
Tetracycline-resistance cassette insertion in VF39SM flaG, Sm r , Tc r
Tetracycline-resistance cassette insertion in 3841 flaG, Sm r , Tc r
3841 strain with mutations in flaA/B/C/D, Sm r , Nm r
VF39SM strain with mutations in flaB/C/D, Sm r , Nm r
VF39SM flaA/B/C/D - complemented with flaA; Sm r , Nm r , Gm r
3841 flaB/C/D -
3841 flaA/B/C/D - complemented with flaA; Sm r , Nm r , Gm r
Cloning vector, Amp r , Km r
Suicide vector with sacB system; Gm r
Suicide vector with sacB system; Gm r
Contains a promoterless gusA-Nm cassette
Contains kanamycin-resistance cassette
Cloning vector, Amp r
pBS::flaD 3'-Km-flaA 5'
flaA 5' fragment (from pCR2.1::flaA 5') subcloned into pBS::flaD 3'-Km, Amp r , Km r
pJQmp18:: flaD 3'-Km-flaA 5'
flaD 3'-Km-flaA 5' fragment subcloned from pBS::flaD 3'-Km-flaA 5' into pJQmp18, Gm r , Km r
Contains omega-spectinomycin cassette; Sp r
Contains tetracycline-resistance cassette; Tc r
Broad-host-range cloning vector, Gm r
pMP220 derivative with promoterless gusA, Tc r
Broad-host-range cloning vector containing flaA gene (with promoter region) from 3841
Broad-host-range cloning vector containing flaA gene (with promoter region) from VF39SM
flaB promoter introduced into pFus1, Tc r
Recombinant DNA techniques
Recombinant DNA techniques were performed using standard methods . Restriction endonucleases used in this study were purchased from Invitrogen or New England Biolabs and used according to the manufacturer's specifications. DNA fragments were isolated from agarose gels using Qiaquick Gel Extraction kit (Qiagen). Plasmids were isolated from E. coli strains using GeneJET™ Plasmid Miniprep kit (Fermentas Life Sciences). Total DNA was isolated from R. leguminosarum strains using Aquapure Genomic DNA Isolation kit (Bio-Rad Laboratories). Primers were synthesized by Sigma Genosys (Sigma-Aldrich) and amplification was carried out using a Multi GeneII PCR machine (Labnet International, Inc.). Southern blots were performed using a non-radioactive technique with reagents and protocols supplied by Roche Applied Science.
Mutagenesis of flagellin genes
The seven fla genes were PCR amplified from R. leguminosarum using the primers listed in Additional file 1. The PCR products were individually cloned into the vector pCR2.1-TOPO using the TOPO Cloning kit (Invitrogen). The genes were excised from the TOPO vector and then ligated into either pJQ200SK or pJQ200mp18 . The details on constructing the individual fla mutants are presented in Additional file 2. Individual mutations in flaA, flaC, flaD, and flaE were introduced by inserting a gusA-Nm r (CAS-GNm) cassette from pCRS530  into the reading frame of each gene. The flaB and flaG genes were mutated by inserting a spectinomycin and tetracycline resistance cassette, respectively, from pHP45:Ω  and pHP45:Ω-Tc . The flaH gene was mutated by inserting a kanamycin-resistance cassette from pBSL99 . The flaA/B/C/D genes were mutated by separately amplifying the 5' end of flaA plus flanking region (missing the 3' end of flaA) and the 3' end of flaD plus flanking region (missing the 5' end of flaD). The truncated genes were cloned separately into pCR2.1-TOPO and the resulting plasmids (pCR2.1::flaA 5' and pBS::flaD 3') were sequenced at the University of Calgary Core DNA Services. The fragment containing the truncated flaD gene was subcloned into pBSIISK+ (Stratagene) creating pBS::flaD 3'. A kanamycin-resistance cassette (Km) from pBSL99  was ligated upstream of the flaD 3' fragment resulting in the construct pBS::flaD 3'-Km. The fragment containing the truncated flaA gene (from pCR2.1::flaA 5') was subcloned into pBS::flaD 3'-Km, upstream of the Km-cassette creating pBS::flaD 3'-Km-flaA 5'. A fragment containing the truncated flaA gene, kanamycin resistance cassette, and truncated flaD gene was subcloned from pBS::flaD3'-Km-flaA 5' into pJQ200mp18  creating pJQmp18::flaD 3'-Km-flaA 5'. Each of the mutated gene/s was introduced into the genome of R. leguminosarum by homologous recombination. The flaA/B/C/D mutants have deletions in the following: flaA 3' end; flaB; flaC; and flaD 5' end. Southern hybridization and/or PCR were performed for each gene to confirm replacement of the wild-type gene with the mutated gene/s.
Construction of gene fusions and ß-glucuronidase (gusA) reporter gene assays
The promoter region of flaB was cloned upstream of a promoterless gusA gene in pFus1 . The resulting construct was introduced into VF39SM and 3841 by biparental mating. VF39SM and 3841 strains containing the flaB-gusA fusion were grown in TY broth for 48 hours at 30°C . β-glucuronidase activity was measured as described by Jefferson et al. and modified by Yost et al.. The data given are the means of triplicate experiments.
Swimming motility test
The strains were grown in TY broth for 24 hours. Swimming motility was determined by inoculating the strains into a motility medium (YES) containing the following: 0.3% agar, 0.01% yeast extract, and 1 mM MgSO4. The optical densities (OD600) of the cultures were standardized and equal amounts of inoculum were inoculated into the swimming agar using a fine-point pipette tip. The swimming diameter was measured 3-4 days after inoculation.
Swarming Motility Test
The swarm assay was performed following the method described by Tambalo et al.. Briefly, R. leguminosarum wildtype and fla mutant strains were grown in TY broth for 24 hours. Equal amounts of inoculum from the TY culture was used to inoculate swarm plates. The plates were incubated at 22°C for two to three weeks and the swarming motility of the fla mutants was compared with the wildtype.
Flagellar filament isolation
Flagellin proteins were isolated from R. leguminosarum based on the procedure described by Maruyama et al.. Cells were grown in 100 ml of TY broth for 48 hours with slow agitation (50 rpm). The bacterial cells were collected by centrifugation at 12,000 × g for 10 minutes. The pellet was resuspended in 40 mM phosphate buffer. The bacterial cells were vigorously agitated using a vortex to detach the flagella from the cells. The mixture was centrifuged at 12,000 × g for 10 minutes using a Sorval centrifuge. The supernatant was removed and centrifuged again at the same speed and time. The supernatant containing the detached flagella was centrifuged in an ultracentrifuge at 50,000 × g for 2 hours. The pellet was resuspended in 200 μL of 40 mM phosphate buffer.
The flagellar protein samples were denatured at 100°C for 5 minutes and then separated on 12% acrylamide SDS-PAGE gel at 200V for 45 minutes. Molecular size markers from Bio-Rad and Fermentas were used. After electrophoresis, the gel was blotted onto a PVDF membrane (Bio-Rad) using the Bio-Rad apparatus and protocol for electrophoretic transfer. The blot was blocked with 10% skim milk solution for 2 hours. After washing with phosphate-buffered saline (PBS) solution, the blot was probed overnight using a polyclonal flagellar antibody raised in a rabbit against isolated flagellar filaments . Protein A-alkaline phosphatase (Sigma-Aldrich) was used as the secondary antibody. The blot was washed with PBS and was developed using NBT/BCIP (Sigma).
Preparation of samples for tandem mass spectrometry analysis (MS/MS)
The flagellar protein samples were run on a polyacrylamide gel as described above. Staining and destaining of the protein gel were performed following standard protocols . The gel was soaked overnight in a staining solution containing 0.1% Coomassie Brilliant Blue (R-250; Sigma), 40% methanol, and 10% acetic acid. Destaining was done using a solution containing 40% methanol and 10% acetic acid. The bands (between approximately 25-37kDa) were excised and submitted to the Southern Alberta Mass Spectrometry (SAMS) Centre at the University of Calgary for LC-MS/MS analysis. Two bands within the size range were observed in the gel. The two bands were analyzed separately for 3841 and in combination for VF39SM.
The gel slices were rinsed once with HPLC-grade water and then twice with 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile. The gel slices were dehydrated with acetonitrile prior to lyophilization. The dehydrated gel was resuspended in 25 mM ammonium bicarbonate (pH8.0) and samples were digested with trypsin. The peptides were extracted from the gel using 1% formic acid in 50% acetonitrile. The extracts were reduced to dryness and then reconstituted in mobile phase of the buffer (3% acetonitrile with 0.2% formic acid) for liquid chromatography.
Tandem mass spectrometry analysis (MS/MS)
The digests were analyzed using an integrated Agilent 1100 LC-Ion-Trap-XCT-Ultra system (Agilent Technologies, Santa Clara, CA), which has an integrated fluidic cartridge for peptide capture, separation, and nano-spraying (HPLC Chip). The injected samples were trapped and desalted for 5 minutes using a pre-column channel (40-nl volume; Zorbax 300 SB-C18) with an auxiliary pump that delivers 3% acetonitrile and 0.2% formic acid at a flowrate of 4 μl/minute. The peptides were reverse-eluted from the trapping column and separated on a 150 mm-long analytical column (Zorbax 300SB-C18) at a flowrate of 0.3 μl/minute. The peptides were eluted using a 5-70% (v/v) acetonitrile gradient in 0.2% (v/v) formic acid over a period of 10 minutes. The MS/MS spectra were collected by data-dependent acquisition, with parent ion scans of 8100 Th/s over m/z 400-2,000. MS/MS scans at the same rate over m/z 100-2200.
Mass Spectrometry Data Analysis
DataAnalysis software for the 6300 series ion trap, v3.4 (build 175) was used to extract the peak-list data. The MS/MS data were analyzed using Mascot v2.1 (Matrix Science, Boston, MA) with the following parameters: 1.6 Da precursor ion mass tolerance, 0.8 Da fragment ion mass tolerance, and one potential missed cleavage. A protein database for R. leguminosarum 3841 was obtained from the Wellcome Trust Sanger Institute website ftp://ftp.sanger.ac.uk/pub/pathogens/rl/ and was deposited in Mascot. The deposited R. leguminosarum 3841 protein database was used for database searching to identify the proteins present in the flagellar preparations. A cut-off score (p = 0.05) of 31 was used for all peptides and since the flagellins of R. leguminosarum are highly homologous, we required at least one unique peptide for a flagellin protein to be considered a match. We also determined the relative abundance of the flagellin proteins based on the exponentially modified protein abundance index (emPAI) values, which were automatically generated using MASCOT analysis. The emPAI value is based on the correlation of the observed flagellin peptides in the MS/MS analysis and the number of observable peptides (obtained by in silico digestion) for each flagellin protein [43, 44].
Flagellar preparations from VF39SM and 3841 were run on 12% acrylamide at 200V for 1 hour and 15 minutes. Glycosylation of flagellin subunits was determined using a Pro-Q Emerald 300 glycoprotein gel stain kit (Molecular Probes) following the manufacturer's instructions. After glycoprotein staining, the total protein was visualized by staining the gel with 0.1% Coommassie Blue.
Transmission electron microscopy
Transmission electron microscopy was performed by slightly modifying the procedure used by Miller et al.. The R. leguminosarum wildtype and fla mutant strains were grown on TY plates at 30°C for 48 hours. A culture suspension was prepared using sterile double distilled water. A formvar carbon-coated grid was placed on top of a cell suspension drop for 3 minutes and excess liquid was removed. Staining was performed using 1% uranyl acetate for 30 seconds. Samples were observed using a Philips 410 transmission electron microscope or a Hitachi-7650 transmission electron microscope with images taken with an AMT Image capture Engine. The length of the flagellar filaments formed by the wildtype and mutant strains was measured using Scion Image http://www.scioncorp.com/.
Results and Discussion
Characterization of flagellin genes in R. leguminosarum
Ultrastructure of the flagellar filament of R. leguminosarum
Transcription of R. leguminosarum fla genes
Previous transcriptional studies in our lab using gusA fusions demonstrated that for both VF39SM and 3841, flaA, flaC, and flaD have the highest expression (2376 Miller Units (MU) to 6516 MU) while minimal expression (68 MU to 542 MU) was observed for flaE, flaH, and flaG. The gene fusion for flaB reported in that paper was made in a different vector, pFAJ1701, so comparisons of flaB expression to that of the other flagellins were not valid. To place levels of flaB transcription in a proper context compared to the other fla genes, a new fusion to the flaB promoter was made in pFus1 (see methods) and gene expression of flaB was measured at 2529 ± 11 MU in 3841 and 4279 ± 466 in VF39SM. These results suggest that flaA, flaB, flaC, and flaD are the major flagellin subunits of R. leguminosarum while flaE, flaH, and flaG play minor roles. However, the presence of post-transcriptional regulation in flagellin biosynthesis cannot be precluded; hence, we performed mutational analysis. We have constructed strains with individual mutations in the seven flagellin genes and two multiple fla mutants (flaB/C/D- and flaA/B/C/D-) for both strains VF39SM and 3841. The resulting mutants were examined for motility defects, using swimming and swarming assays, and morphological defects, using transmission electron microscopy.
Motility assays and electron microscopy of wildtype and fla mutant strains
Properties of R. leguminosarum wildtype and flagellin mutants
Effective Fla subunit(s)
Normal (4.7 ± 0.5um; n = 8)
Almost all cells are non-flagellated; only one cell with very thin, short appendage
Truncated (2.2 ± 0.5um; n = 6)
Truncated (3.4 ± 0.3 um; n = 5)
Truncated (2.4 ± 0.6 um; n = 12)
Truncated (1.9 ± 0.6 um; n = 13)
Normal (5.1 ± 0.5 um; n = 13)
Truncated (1.6 ± 0.5 um; n = 6); reduced number of filaments (1-2 filaments/cell)
Truncated (2.1 ± 0.5 um; n = 9); reduced number of filaments (1-2 filaments/cell)
Normal number and length; thinner filaments
Normal; slightly reduced number of filaments
Truncated (1.6 ± 0.3 um; n = 13); reduced number of filaments (1-2 filaments/cell)
Flagellin subunits and their relative abundance in R. leguminosarum wildtype strains based on tandem mass spectrometry analysis.
No. of unique peptides detected
Sequence coverage (%)
A. 3841 wt lower band
B. 3841 wt upper band
A. VF39SM wt
The motility assays and the filament morphologies demonstrate that FlaA is an essential flagellin subunit for R. leguminosarum. Mutation of flaA resulted in non-flagellated (for VF39SM) and consequently non-motile strains. It is possible that (at least for strain VF39SM), FlaA forms the proximal part of the filament, hence when FlaA is not synthesized, R. leguminosarum fails to assemble the distal part of the filaments using the other subunits synthesized. The major role of FlaA in filament assembly and function is similar to what has been reported in S. meliloti, A. tumefaciens, and R. lupini[5, 6]. In all three species, mutation of flaA resulted in non-motile strains. However, unlike the non-flagellated VF39SM flaA mutant, strains of S. meliloti, A. tumefaciens and R. lupini with mutations in flaA were able to polymerize severely truncated filaments. Whereas FlaA is an essential subunit, it is not sufficient to assemble a fully functional flagellar filament as demonstrated in the flaB/C/D mutants. The flaB/C/D mutant strains exhibited shorter filaments and have reduced numbers of flagella (Table 2), which might have been assembled using FlaA and the other minor flagellin subunits (FlaE/H/G). In addition, the assembled filaments were not fully functional as demonstrated by the motility assays.
It is also apparent from our functional studies that both FlaB and FlaC are major components of the flagellar filament since mutation in each of the genes resulted in shorter filaments, reduced number of flagella, and consequently reduced motility. It is possible that FlaB and FlaC are located in the middle part of the filament, hence only the proximal part of the filament, composed of FlaA and possibly other minor subunits, is formed in the flaB and flaC mutants. Additionally, the reduction in the length and number of filaments in the flaB and flaC mutants may reflect an increase in the brittleness and fragility of the filament. Our claim that FlaA, FlaB, and FlaC are the major flagellins of VF39SM and 3841 is further supported by our gene expression studies which demonstrated high promoter activities for flaA, flaB, and flaC. It is also possible that FlaD contributes to the flagellar filament since the amount of flaD transcript was also high and the filaments formed by the VF39SM flaD mutant were thinner than the wildtype. The formation of thinner filaments also suggests that FlaD might be located along the entire length of the filament for VF39SM, thus the need for a high amount of flaD transcripts. However, it is remarkable that the swimming and swarming motility of the VF39SM flaD mutant are not impaired. A possible explanation could be that the width of the filament formed by the flaD mutant is still enough to support the normal function of the flagella. Contrary to the major roles of FlaA/B/C/D in VF39SM, FlaE, FlaH, and FlaG appear to be minor components of the flagellar filament as indicated by expression levels as measured in gene fusions, and by the subtle effects of their mutations on flagellar filament morphology and on motility. In 3841, FlaE and FlaH appeared to be important for swimming but not for swarming motility. Since the TEM images for the wildtype and fla mutant strains were obtained from vegetative cells, it would be interesting to observe the filaments formed by the swarm cells of 3841 flaE and 3841 flaH mutants.
Tandem mass spectrometry analysis
The locations of the flagellin peptides detected in the flagellar preparations are indicated in Fig. 1 and 2. Only FlaA, FlaB, and FlaC peptides were detected in the flagellar preparation for strain 3841 (for both the lower and the upper bands; Table 3) with sequence coverage ranging from 31% to 46%. These three subunits also comprised the majority of the flagellin subunits detected in VF39SM (Table 3). FlaE and FlaG comprised a small fraction of the flagellin subunits detected in the VF39SM wt strain. The sequence coverage for the flagellin subunits detected in VF39SM ranged from 18% to 46%. The results obtained from the MS/MS analysis indicate that at least three flagellin subunits (FlaA/B/C) are incorporated into the functional flagellar filament of strain 3841 while VF39SM polymerizes five flagellins (FlaA/B/C/E/G) into its flagellar filament. The consistently shorter flagellar filaments formed by the flagellin mutants (VF39SM/3841 flaB and flaC mutants) and the absence of flagellar filaments in VF39SM flaA mutants and nearly all cells of 3841 flaA- also suggest that the major subunits (FlaA, FlaB, and FlaC), at least, are present in the complete flagella that are assembled.
Peptides for FlaD, FlaE, FlaH, and FlaG were not detected in the flagellar preparation for 3841 while FlaD peptides were not detected in VF39SM. The absence of the flagellin subunits could be due to the following reasons: (a) they are not synthesized under the conditions tested; (b) the subunits are synthesized but at a very low concentration, hence they remained undetected; and/or (c) the flagellin subunits are highly unstable. For strain 3841, mutation of flaE and flaH resulted in a reduction in swimming motility, suggesting that these subunits probably contribute to the flagellar filament. However, FlaE and FlaH peptides were not detected in the wildtype flagellar preparations, indicating that these peptides may not be stable under the conditions used.
Glycosylation of flagellin subunits
We observed that for strain 3841, both the upper and the lower bands on the protein gel contained the same set of flagellin subunits (FlaA, FlaB, and FlaC) (Table 3). The molecular masses (around 35kDa; Additional file 3) of the bands observed on the gel also appeared to be higher than the predicted molecular masses (31kDa) for FlaA and FlaB. This suggests that at least FlaA and FlaB may have undergone post-translational modification, resulting in a higher molecular weight and subsequently slower migration in the protein gel.
Analysis of the flagellin amino acid sequences of R. leguminosarum (Fig. 1 &2) revealed the presence of two to four putative glycosylation signals (N-X-S/T, where X is any amino acid except proline) . The MS/MS spectral data for the identified peptides containing the glyosylation signal were also analyzed for the presence of glycosylation, based on the presence of peaks (m/z) corresponding to different types of glycosylation (Additional file 4 shows a sample of a MS/MS spectrum). However, we have not identified any potential glycosylation for these peptides which may be attributed to the lability of this modification [56, 57]. Also, sequence coverage only ranged from 18% to 46% (Fig. 1 and 2) and peptides at the C-termini of the flagellin subunits were not detected. The C-terminus contains a common glycosylation site for the R. leguminosarum flagellin subunits but these glycosylations were not detected in the MS/MS analysis, which could be due to the above reason. Thus, we performed glycoprotein staining to determine if the flagellins are post-translationally modified by glycosylation. We observed positive staining for the flagellins of both VF39SM and 3841 suggesting that these flagellins are glycosylated (Fig. 6). We were unable to determine which flagellins are glycosylated because the seven flagellins were not separated on the protein gel. Glycosylation of flagellins has been reported in a number of animal and plant pathogens including Campylobacter jejuni[56, 57], Helicobacter pylori[57, 58], Pseudomonas aeruginosa[59, 60], Pseudomonas syringae[61, 62], Listeria monocytogenes[63, 64], A. tumefaciens, Acidovorax avenae, as well as in the nitrogen-fixing bacterium Azosprillum brasilense. It has been suggested that glycosylation may play a role in flagellar filament assembly and in pathogenesis [67, 68]. In soil bacteria, it may also function in the attachment of bacteria to the plant roots , and in avoiding recognition by the host plant .
In this study, we were able to clarify the roles of the seven flagellin subunits in the assembly of the flagellar filament in R. leguminosarum. Taken altogether, our results indicate that FlaA is an essential subunit, but that it is not enough to assemble a fully functional flagellar filament. FlaB and FlaC are major components of the filament while FlaD, FlaE, FlaH, and FlaG are only minor components. To assemble a fully functional filament, at least three (FlaA, FlaB, and FlaC) and five (FlaA, FlaB, FlaC, FlaE, and FlaG) flagellin subunits should be synthesized by 3841 and VF39SM, respectively. There were no substantial differences in the requirements for individual flagellins in swimming vs. swarming motility. The flagellins of 3841 and VF39SM are possibly modified by glycosylation.
List of Abbreviations
tandem mass spectrometry
exponentially modified protein abundance index
transmission electron microscopy
Sodium-dodecyl sulfate polyacrylamide gel electrophoresis.
We gratefully acknowledge the support for this work from Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to MFH and SFK. DDT was supported by a Government of Canada graduate scholarship and the Bettina Bahlsen scholarship. We thank Carol Stremick for her help with the protein work as well as Wei-Xiang Dong at the Microscopy and Imaging Facility of the University of Calgary for his assistance with electron microscopy.
We also thank Dr. Christopher K. Yost for his very helpful comments on the manuscript.
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