Effects of Calcium and Signal Sensing Systems on Azorhizobium caulinodans Biofilm Formation and Host Colonization

Abstract

Biofilm formation is important for establishing plants-microbe associations. The role of calcium on biofilm formation has been studied in many bacteria except rhizobia. In this study, we investigated the role of calcium for biofilm formation in Azorhizobium caulindans, which forms nodules in the stem and root of its host plant Sesbania rostrata. We found that calcium is essential for A. caulindans biofilm formation, in addition to the presence of extracellular matrix components, eDNA and proteins. Also, calcium-mediated biofilm formation was tested with chemotaxis, motility, cyclic di-GMP synthesis, and quorum sensing mutants. Finally, calcium was found to promote S. rostrata root colonization of A. caulinodans. In total, these results show that calcium is essential for A. caulindans biofilm formation, and it affects the interaction between A. caulinodans and host plant.

Keywords: Azorhizobium caulinodans; biofilm; calcium; chemotaxis; eDNA; quorum sensing.

FIGURE 1

Biofilm formation of Azorhizobium caulinodans at various timepoints with different media components. (A) Biofilm formation of A. caulinodans with TY or L3 medium. Representative images of A. caulinodans biofilm on the wall of glass tubes after culturing for 5 days. (B) A. caulinodans biofilm formation in TY medium from 0.5 to 12 h. Images on the top are representative biofilms on the wall of 96-well plates. (C) Biofilm formation of A. caulinodans after 6, 9, and 12 h, when different components were removed from TY medium. Values are shown as the means and standard deviations from at least three independent experiments. The same letter above the error bars indicates not statistically different with results after culturing for 0.5 h (B) or under same conditions (C) by a Duncan’s test. Asterisks (^∗) means P < 0.05 vs. the results after same time with TY medium by a Student’s t test.

FIGURE 2

Biofilm formation of A. caulinodans with different concentrations of calcium and calcium chelator. (A) Calcium dose (0 to 900 mM) response of A. caulinodans biofilm formation with TY medium after 6, 9, and 12 h. Below the x axis means control without adding bacteria. Images on the top show the biofilm formed on the wall of 96-well plates. (B) Biofilm of A. caulinodans with different concentrations of calcium on L3 minimal medium after 6 and 12 h. (C) Biofilm for A. caulinodans with TY adding different concentration of calcium chelator, EGTA. Values are shown as the means and standard deviations from at least three independent experiments. The same letter above the error bars indicates not statistically different with results under same treatment by a Duncan’s test. Asterisks (^∗) and (^∗∗) mean P < 0.05 and P < 0.01 vs. the results after culturing same time with 0 mM calcium (A) or TY medium (C) by a Student’s t test.

FIGURE 3

The roles of exopolysaccharides, eDNA, and extra cellular proteins on biofilm formation of A. caulinodans. (A) Biofilm formation of wild type, Δazc_1831 and Δoac after 6 and 12 h with TY medium. (B) After 9 h, the biofilm biomass of wild type after adding DNase or Proteinase K from 0 to 2 h. Values are shown as the means and standard deviations from at least three independent experiments. The same letter above the error bars indicates not statistically different with results after culturing for same time (A) or same treatment (B) by a Turkey HSD’s test. Asterisks (^∗∗) means P < 0.01 vs. the wild type after culturing for same time by a Student’s t test.

FIGURE 4

Biofilm formation of wild type and different chemotaxis and motility defective mutants. (A) Biofilm formation of chemotaxis defective mutants ΔcheA, ΔcheA-R, ΔcheZ, ΔcheY1, ΔcheY2, Δazc_0821, and Δazc_0660 with TY medium after 6 and 12 h. (B) Two motility defective mutants ΔfliN and ΔmotA form biofilm with TY medium after 6, 9, and 12 h. The same letter above the error bars indicates not statistically different with results under same culturing time by a Duncan’s test. (C) Swimming behavior of A. caulinodans wild type and ΔcheA on soft-agar plates with or without 6 mM calcium. Asterisks (^∗P < 0.05; ^∗∗P < 0.01) show a significant difference between conditions according to a t-test. Values are shown as the means and standard deviations from at least three independent experiments.

FIGURE 5

The role of c-di-GMP and quorum sensing on biofilm formation. (A) Biofilm formation of c-di-GMP increased and decreased mutants, Δazc_0308 and Δazc_2412 after 6, 9, and 12 h. (B) Biofilm formation of wild type with different initial bacterial concentrations from OD₆₀₀ of 0.25 to 2.5 after 6 and 9 h. (C,D) The relationship between the biofilm biomass and initial bacterial concentration for different quorum sensing luxR-like gene mutants, Δazc_0572, Δazc_2281, Δazc_3095, Δazc_4108 after 6 h (C) and 9 h (D). Values are shown as the means and standard deviations from at least three independent experiments. The same letter above the error bars indicates not statistically different with results under same culturing time by a Duncan’s test.

FIGURE 6

Colonization of wild type on S. rostrata roots with L3 or TY medium including calcium or not. The concentration of calcium in L3 and TY mediums is 0.36 and 6 mM, respectively. Values are shown as the means and standard deviations from at least three independent experiments. Asterisks (^∗) means p < 0.05 vs. each medium without calcium by a Student’s t test.