Figure 1.
Tropolone production by B. plantarii in the coculture system.
Tropolone production was quantified in the mono-culture of B. plantarii (red circle), and in the co-culture system of B. plantarii and T. virens PS1-7 (blue triangle). Values are means ± SD (shown as error bars) (n = 3).
Figure 2.
Active principle from T. virens PS1-7 for repression of tropolone production by B. plantarii.
Tropolone production was semi-quantified by the density of dark crystallines formed by chelation of B. plantarii-produced tropolone with iron supplemented to the medium at 0.1 mM. Repression of tropolone production was observed in the area around the paper disc charged with solvent (A), the area around the paper disc charged with fraction 2 equivalent to 3 ml culture fluid (35 µg disc−1) (B), and with fraction 3 equivalent to 3 ml culture fluid (55 µg disc−1) (C). Red arrow indicates the typical tropolone-iron crystallines. Major component in the fractions 2 and 3 were identical with carot-4-en-9,10-diol. Its chemical structure including the relative configuration was shown in this figure.
Figure 3.
Virulence-attenuation effect of carot-4-en-9,10-diol on the growth of the rice seedlings inoculated with B. plantarii.
Typical root and shoot growth performance among rice seedlings inoculated with B. plantarii (control, A), B. plantarii-inoculated rice seedlings that were also treated with 20 µM carot-4-en-9,10-diol at the same time (treated, B), inoculated rice seedlings without any inoculation of B. plantarii (blank, C). Virulence of B. plantarii recorded as the shoot and the root growth inhibition as indexes of the symptom was attenuated with statistical significance (D, right panel). Values are means ± SD (shown as error bar) (n = 30). *P<0.01 by Student’s-t test.
Figure 4.
Effect of carot-4-en-9,10-diol on B. plantarii tropolone production.
(A) Tropolone production was quantified from cultures of B. plantarii in PDB containing carot-4-en-9,10-diol at 20 µM (blue triangle), 200 µM (green diamond), and in the PDB without carot-4-en-9,10-diol (red circle). (B) Tropolone was analyzed quantitatively at 72 h for culture medium inoculated with B. plantarii containing carot-4-en-9,10-diol at zero, 10, 20, 50, 100 and 200 µM. Values are means ± SD (shown as error bar) (n = 3).
Figure 5.
Effect of carot-4-en-9,10-diol on B. plantarii cell growth and morphology.
(A) Cell growth was quantified from B. plantarii PDB cultures containing carot-4-en-9,10-diol at 20 µM (blue triangle), 200 µM (green diamond), and in PDB without carot-4-en-9,10-diol (red circle). Values are means ± SD (shown as error bars) (n = 3). (B) Cell morphology was observed at 30 h for culture medium inoculated with B. plantarii containing no carot-4-en-9,10-diol (left panel, control) and 20 µM (right panel). Yellow arrowheads indicate typical cell aggregation.
Figure 6.
Relationship between biofilm formation and tropolone production in B. plantarii.
Correlation analysis of biofilm biomass and endogenous tropolone produced by B. plantarii incubated for 24, 36, 48, 72, 84 and 96 h in the static culture system. The endogenous tropolone production showed a positive and linear correlation with the biofilm formation evaluated by crystal violet staining method with the correlation coefficient of r2 = 0.96 (n = 18). Both tropolone production and biofilm formation increased along with longer incubation time up to 96 h. Values shown by the plots are means ± SD (n = 3). Vertical and horizontal error bars on each plot were for the biofilm biomass (OD570) and the tropolone production (mM) respectively.
Figure 7.
Morphological and physiological characteristics of biofilms formed by B. plantarii exposed to tropolone or carot-4-en-9,10-diol.
(A) General state of B. plantarii biofilm formation after a 48 h incubation in PDB containing 200 µM exogenous tropolone only (top, right panel), 200 µM exogenous tropolone plus 200 µM carot-4-en-9,10-diol (bottom, left panel), 200 µM carot-4-en-9,10-diol (bottom, right panel), and in PDB containing neither exogenous tropolone nor carot-4-en-9,10-diol as control (top, left panel). The microscopic observation was done with a 10× objective lens, after the cells were stained by a crystal violet. (B) Three dimensional structures of representative cell aggregates in each treatment after 48 h incubation were also observed under a phase contrast mode, with the same relative positions of each panel with that of A. Red arrows indicate the biofilm induced by tropolone, while yellow arrows indicates the biofilm induced by carot-4-en-9,10-diol. (C) Comparison of matrix of the biofilm formed by B. plantarii incubated for 96 h with endogenous tropolone (top panel, control) or 20 µM carot-4-en-9,10-diol (bottom panel). Black arrow indicates the typical fibrous matrix.
Figure 8.
Cell viability assay for the biofilm induced by tropolone or carot-4-en-9,10-diol.
Comparison of cell viability of the biofilms formed by B. plantarii that was incubated for 96 h in response to endogenous tropolone (control) or supplementation of 10 µM carot-4-en-9,10-diol was done by observation of fluorescently-labelled biofilms (A, left panels) and quantification of living/dead cells (B, right columns). In panels in A, cells showing green fluorescence are living cells, while red are dead. Values are means ± SD (shown as error bars) (n = 3). *P<0.01 by Student’s-t test.
Figure 9.
Quantitative real time PCR analysis of the effects of carot-4-en-9,10-diol and quorum sensing inhibitors on plaI (A) and plaR (B) gene expression in B. plantarii.
B. plantarii was incubated in PDB containing 20 µM carot-4-en-9,10-diol, 50 µM 3-methyl-2(5H)-furanone, or 100 µM DMDS. Control as PDB containing solvent only. Values are means ± SD (shown as error bars) (n = 3). *P<0.01, **P<0.001 by Student’s-t test.