Table 1.
Resistance phenotypes: GmR, KmR, NxR, ApR and TcR, gentamicin, kanamycin, nalidixic acid, ampicillin and tetracycline, respectively.
Table 2.
Plant responses to inoculation of Glycine max cv. Osumi with S. fredii SMH12 and derivatives.
Table 3.
Glycine max cv. Osumi root attachment.
Figure 1.
Visualization of the soybean root colonization.
A. Epifluorescence microscopy analysis of the colonization of the soybean rhizosphere by gfp-tagged bacteria [SMH12, SVQ648, SMH12 (pME6863)]. Roots were visualized 7 days after inoculation. 1. Proximal root. 2. Lateral roots. Bar, 100 µm. B. Scanning microscopy analysis of the colonization of the soybean rhizosphere by SMH12, SVQ648 and SMH12 (pME6863). Roots were visualized 7 days after inoculation. 1. Proximal root. 2. Lateral roots. Bar, 5 µm. SMH12: wild-type, SVQ648: nodD1 mutant, SMH12 (pME6863): lactonase strain.
Figure 2.
Biofilm structure of S. fredii SMH12 and derivatives on glass surfaces: reconstruction of the Z-stacks and measure of the surface coverage.
Main fluorescence value of the wild-type strain was arbitrarily given a value of 100. Averages and standard deviations of five randomized optical fields per strain corresponding to two independent experiments are shown. The asterisks indicate a significant different at the level α = 5% with respect to wild-type strain by using the Mann-Whitney non-parametrical test. Left side corresponds to cultures without flavonoids. Right side corresponds to cultures with inducing flavonoid. A. SMH12. B. SVQ648. C. SMH12 (pME6863). D. SMH12 (pME6000). Bar, 20 µm. SMH12: wild-type, SVQ648: nodD1 mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.
Figure 3.
Biofilm structure of S. fredii SMH12 and derivatives on glass surfaces: reconstruction of the XY-axis, XZ-axis and YZ-axis. The top corresponds to cultures without flavonoids.
The bottom corresponds to cultures with inducing flavonoid. A. SMH12. B. SVQ648. C. SMH12 (pME6863). D. SMH12 (pME6000). Bar, 20 µm. SMH12: wild-type, SVQ648: nodD1 mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.
Table 4.
β-galactosidase activity obtained using an adapted assay with A. tumefaciens NT1 (pZLR4) as bioreporter and grown in the presence of supernatants from biofilm cultures (1% v/v).
Figure 4.
Quantitative RT-PCR analysis of the expression of traI and nodA from S. fredii SMH12 and derivatives from biofilm cultures.
Expression data shown are the mean (± standard deviation of the mean) for three biological replicates. Expression was calculated relative to the expression without flavonoids of the wild-type strain by using the Mann-Whitney non-parametrical test. The asterisks indicate a significant different at the level α = 5%. White bars: biofilm cultures without flavonoids. Gray bars: biofilm cultures with genistein. A. traI relative expression. B. nodA relative expression. SMH12: wild-type, SVQ648: nodD1 mutant, SMH12 (pME6863): lactonase strain.
Figure 5.
Adhesion of S. fredii SMH12 and derivatives on polystyrene surfaces.
Biofilms were measured as the amount of crystal violet absorbed by the biofilm formed on multi-well plates and determined by absorbance at 570 nm after de-staining with ethanol (see methods). Absorbance of the wild-type strain was arbitrarily given a value of 1. Averages and standard deviations of eight replicas per strain corresponding to five independent experiments are shown. The asterisks indicate a significant different at the level α = 5% with respect to wild-type strain by using the Mann-Whitney non-parametrical test. A. Dark gray bars correspond to experiments performed without flavonoids, white to experiments with umbelliferone and light grey bars to experiments with genistein. B. Dark gray bars correspond to experiments performed without flavonoids and light grey bars to experiments with genistein. 3-oxo-C8-HSL and C8-HSL are used at 5.5 µM. C14-HSL is used at 55 µM. SMH12: wild-type, SVQ648: nodD1 mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.
Figure 6.
Model of biofilm formation in S. fredii SMH12.
A. In soil. B. In rizosphere. S. fredii SMH12 forms monolayer-type biofilm when colonize soil surfaces. When the bacterium colonizes the legume root (in presence of inducing flavonoids), it forms microcolony-type biofilm, which is necessary for a successful root colonization and symbiosis between rhizobia and legume.