Inhibition of Agrobacterium tumefaciens Growth and Biofilm Formation by Tannic Acid

Abstract

Agrobacterium tumefaciens underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important plant species. The bacterium initiates infection by colonizing and forming biofilms on plant surfaces, and thus, novel compounds are required to prevent its growth and biofilm formation. In this study, we investigated the ability of tannic acid, which is ubiquitously present in woody plants, to specifically inhibit the growth and biofilm formation of A. tumefaciens. Tannic acid showed antibacterial activity and significantly reduced the biofilm formation on polystyrene and on the roots of Raphanus sativus as determined by 3D bright-field and scanning electron microscopy (SEM) images. Furthermore, tannic acid dose-dependently reduced the virulence features of A. tumefaciens, which are swimming motility, exopolysaccharide production, protease production, and cell surface hydrophobicity. Transcriptional analysis of cells (Abs600 nm = 1.0) incubated with tannic acid for 24 h at 30 °C showed tannic acid most significantly downregulated the exoR gene, which is required for adhesion to surfaces. Tannic acid at 100 or 200 µg/mL limited the iron supply to A. tumefaciens and similarly reduced the biofilm formation to that performed by 0.1 mM EDTA. Notably, tannic acid did not significantly affect R. sativus germination even at 400 µg/mL. The findings of this study suggest that tannic acid has the potential to prevent growth and biofilm formation by A. tumefaciens and thus infections resulting from A. tumefaciens colonization.

Keywords: Agrobacterium tumefaciens; biofilm; quantitative reverse transcription PCR; tannic acid; virulence.

Figure 1

Tannic acid is composed of a central glucose with ten surrounding galloyl units (A). Biofilm formation of A. tumefaciens on polystyrene (B). Gross images and biofilm morphology on 6-well polystyrene plates (C). The normalized ratio of biofilm (Abs570 nm) to cell growth (Abs620 nm) shows tannic acid reduces biofilm formation at ≥150 µg/mL (D). Cells were incubated in LB containing tannic acid at the concentrations shown for 48 h at 30 °C under static conditions. Two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. Asterisks (*) denote significant differences (p ≤ 0.05 by the two-tailed t-test) between treated groups and nontreated controls.

Figure 2

The 3D-thermal LUT plots demonstrating concentration-dependent biofilm formation on flat polystyrene surfaces by nontreated and tannin-treated A. tumefaciens. Biofilms were formed on polystyrene surface in 6-well plates in LB with or without tannic acid for 48 h at 30 °C under static conditions. Crystal violet (0.1%) stained biofilms (for 20 min) were imaged using iRisTM Digital Cell imaging, Logos Biosystems, Annandale, VA, USA. The color-coded pictures correspond to the scale (right side of the image) where zero shows negligible biofilm and 240 represents maximum formation of biofilm. Two independent bacterial colonies were used in triplicates (n = 6), and experiments were repeated twice.

Figure 3

Growth inhibition of A. tumefaciens by tannic acid cultured in LB for 24 h at 30 °C. Growth curves (A) and percent cell viability (B) after exposure to tannic acid. Two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. ‘*’ denotes significant difference between nontreated and treated A. tumefaciens. p ≤ 0.05 as determined by the two-tailed t-test.

Figure 4

Effect of iron limitation on growth and biofilm formation of A. tumefaciens. Line and scatter plots represent cell growth absorbance at 620 nm and bars show the amount of biofilm produced (570 nm) in 96-well microtiter plates after 48 h incubation at 30 °C in LB. Effect of increasing concentrations of iron source (Fe₂SO₄·7H₂O) with constant EDTA (1 mM) (panel (A)), constant 100 µg/mL tannic acid (panel (B)), and 200 µg/mL tannic acid (panel (C)). Bar for ‘none’ shows biofilm formation without EDTA or tannic acid or Fe₂SO₄·7H₂O. Biofilm (570 nm) bars marked with ‘†’ show biofilm in the presence of an iron source. Two independent bacterial colonies were used in triplicates (n = 6), and each experiment was repeated at least twice. Asterisks denote significant differences between ‘none’ and treated A. tumefaciens at p ≤ 0.05 by the two-tailed t-test.

Figure 5

Tannic acid concentration-dependent decrease in the virulence traits of A. tumefaciens. Exopolysaccharides (EPS) production (A), protease activity (B), and percent cell surface hydrophobicity (%CSH) (C) after incubation at 250 rpm for 24 h at 30 °C in LB. Concentration-dependent effect of tannic acid on the swimming motility of A. tumefaciens (D) after incubation for 48–72 h tannic acid containing peptone agar (1% peptone, 0.5% NaCl, 0.25% agarose). Swim diameters were recorded after incubation for 48 or 72 h (E). For EPS production, protease activity, and %CSH, two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. For swimming motility, three independent colonies were used (n = 3), and experiment was repeated twice. Asterisks denote significant differences between nontreated and treated A. tumefaciens. p ≤ 0.05 by the two-tailed t-test.

Figure 6

A. tumefaciens biofilm formation (48 h at 30 °C in LB) by nontreated cells (A–C) and tannic acid (200 µg/mL) treated cells (D–F) on nitrocellulose membranes. Biofilm formation by A. tumefaciens on the roots of R. sativus in the absence (G–I) and presence of tannic acid at 200 µg/mL (J–L) as visualized by scanning electron microscopy (SEM) at different magnifications. Two independent bacterial colonies were used in triplicates (n = 6), and at least five micrographs captured from five different random locations for each sample were analyzed.

Figure 7

Induction of tumors by A. tumefaciens on potato discs and its inhibition by tannic acid (200 µg/mL). Bars show numbers of tumors per disc while symbols represent tumor induction (%). Representative pictures of potato discs with tumors marked with blue arrows are shown on the top. Two independent bacterial colonies were used in triplicates (n = 6), and experiment was repeated twice. ‘*’ shows a significant difference at p ≤ 0.05 by the two-tailed t-test.

Figure 8

Toxicity assessment of tannic acid on R. sativus seed germination over 5 days on tannic acid infused into 0.7% agar and 0.86 g/L MS medium (A). Bar plot of percentage seed germination as a function of increasing tannic acid concentration (B). Seeds (n = 10) were used for each concentration, and experiment was repeated twice.

Figure 9

qRT-PCR analysis of the expressions of genes after treating A. tumefaciens with tannic acid at 200 µg/mL. The asterisk denotes a significant difference between nontreated and treated A. tumefaciens. Two independent bacterial colonies were used in duplicates (n = 4), and experiment was repeated twice. p ≤ 0.05 by the two-tailed t-test.