Divalent Cations (Ca2+, Mg2+, Mn2+, Fe2+, Ni2+, and Zn2+) Enhance Growth of Xanthomonas citri and X. campestris by Reducing Generation Time

Abstract

Bacterial ion homeostasis is critical for adaptation and survival. In this study, we show that supplementing 2xTY medium with divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ni²⁺, and Zn²⁺) from 50 to 1,000 μM significantly enhances the growth of Xanthomonas species by reducing generation time, while monovalent ions (Na⁺, K⁺, Li⁺, and Cl^-) had no significant effect. Among them, Ca²⁺ effectively boosted X. citri fitness, increasing the specific growth rate by up to 223%, shortening the cell cycle, and enhancing adhesion to abiotic surfaces up to 8-fold. Calcium also induced a more uniform and smaller cell morphology, suggesting improved cell division and metabolic efficiency. Colony forming unit counts confirmed a higher viability, excluding dead cell accumulation. These effects were conserved in other Xanthomonadaceae members (X. campestris and S. maltophilia) but not in nonrelated species. Based on these findings, we propose an optimized growth medium named 2xTY-Ca (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 1 mM CaCl₂), which significantly improves Xanthomonas spp. growth kinetics in vitro, enhancing X. citri and X. campestris cultivation for laboratory and industrial applications. Though the precise molecular mechanism behind these phenotypes has yet to be determined, we highlight X. citri as a promising organism to understand the role of divalent cations in bacterial physiology.

1

X. citri growth curves in the presence of monovalent and divalent cations. Growth curves of X. citri pv. citri 306 WT in 2xYTON medium supplemented with different concentrations of cations, ranging from 100 nM to 10 mM for NaCl (A), KCl (B), and LiCl (C), and from 50 to 1,000 μM for CaCl₂ (D), MgCl₂ (E), MnCl₂ (F), FeCl₂ (G), NiCl₂ (H), ZnCl₂ (I), and CoCl₂ (J). Growth curve of X. citri pv. citri 306 grown in 5 mL of 2xTY or 2xYTON medium with and without supplementation by 5 mM CaCl₂ (K). Three experimental replicates were performed.

2

X. citri CFU counting in the presence of monovalent and divalent cations. Overnight cultures were used for inoculation in each condition and for the control group (without ions). CFU counting of X. citri pv. citri 306 wild type incubated in 5 mL of 2xYTON medium with or without ion supplementation. The bacteria were incubated for an additional 12 h before plating in 2xYTON agar for CFU quantification. Cations were added in concentrations of 5 mM for NaCl (A), KCl (B), LiCl (C), CaCl₂ (D), MgCl₂ (F), and MnCl₂ (G), and 1 mM for FeCl₂ (H), NiCl₂ (I), ZnCl₂ (J), and CoCl₂ (K). CFU counting of X. citri pv. citri 306 ΔgumD was performed without and with 5 mM CaCl₂ (E). Three experimental replicates were performed. Two biological replicates (n = 3) were performed for the CoCl₂ condition.

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X. citri growth stabilization. (A) Influence of different concentrations of NaCl on the growth of X. citri pv. citri 306. The dark arrow indicates the drop from the highest to the lowest absorbance values after the exponential growth phase. (B) The combined effect of 5 mM CaCl₂ with varying NaCl concentrations on X. citri growth. The dashed black square highlights the prevention of the absorbance decline in cultures grown with 5 mM CaCl₂ jointly with 50, 100, or 250 mM NaCl. Arrows, colored according to the curve scheme, indicate the highest and lowest absorbance values reached under each condition after entering the stationary growth phase. Five experimental replicates were performed.

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Impact of monovalent and divalent cations on the morphology of X. citri. X. citri pv. citri 306 cells were incubated for 12 h in 2xYTON medium without ion addition (A) and in 2xYTON medium supplemented with 5 mM NaCl (B), 5 mM KCl (C), 5 mM CaCl₂ (D), 5 mM MgCl₂ (E), and 5 mM MnCl₂ (F). Negative staining was performed using a 2% uranyl acetate solution. 100 cells were used to calculate the parameters shown in panels (A–F). Scale bar = 1 μm.

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Effects of CaCl₂, NaCl, and EGTA on the growth of bacterial species from different phyla. Growth curves of Staphylococcus aureus ATCC 25923 (A), Acinetobacter baumannii ATCC 19606 (B), Stenotrophomonas maltophilia K279a (C), and Escherichia coli K-12 (D) in 2xYTON medium, Caulobacter crescentus NA1000 (E) in PYE (complete) and PYE* (without CaCl₂ and MgCl₂) media, and Leptospira biflexa sv. Patoc (Ames) in EMJH medium supplemented with Leptospira Enrichment EMJH (F). A total of three experimental replicates were performed.

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The effect of supplementation of calcium on the growth medium of different bacteria in the polystyrene adhesion microplate. Calcium importance for polystyrene adhesion in the microplate of Xanthomonas citri pv. citri 306 (A), Escherichia coli K-12 (B), Acinetobacter baumannii ATCC 19606 (C), and Staphylococcus aureus ATCC 25923 (D). X. citri was incubated at 28 °C for 5 days, while E. coli, A. baumannii, and S. aureus were incubated at 37 °C for 48 h, all in static conditions. *p < 0.05, **p < 0.005, and ***p < 0.0005. A total of three experimental replicates were performed.