A membrane protein of the rice pathogen Burkholderia glumae required for oxalic acid secretion and quorum sensing

Abstract

Bacterial panicle blight is caused by Burkholderia glumae and results in damage to rice crops worldwide. Virulence of B. glumae requires quorum sensing (QS)-dependent synthesis and export of toxoflavin, responsible for much of the damage to rice. The DedA family is a conserved membrane protein family found in all bacterial species. B. glumae possesses a member of the DedA family, named DbcA, which we previously showed is required for toxoflavin secretion and virulence in a rice model of infection. B. glumae secretes oxalic acid as a "common good" in a QS-dependent manner to combat toxic alkalinization of the growth medium during the stationary phase. Here, we show that B. glumae ΔdbcA fails to secrete oxalic acid, leading to alkaline toxicity and sensitivity to divalent cations, suggesting a role for DbcA in oxalic acid secretion. B. glumae ΔdbcA accumulated less acyl-homoserine lactone (AHL) QS signalling molecules as the bacteria entered the stationary phase, probably due to nonenzymatic inactivation of AHL at alkaline pH. Transcription of toxoflavin and oxalic acid operons was down-regulated in ΔdbcA. Alteration of the proton motive force with sodium bicarbonate also reduced oxalic acid secretion and expression of QS-dependent genes. Overall, the data show that DbcA is required for oxalic acid secretion in a proton motive force-dependent manner, which is critical for QS of B. glumae. Moreover, this study supports the idea that sodium bicarbonate may serve as a chemical for treatment of bacterial panicle blight.

Keywords: bacterial panicle blight; oxalic acid; pH homeostasis; quorum sensing.

FIGURE 1

Growth and culture medium pH of Burkholderia glumae wild type (336gr‐1), ΔdbcA, and ΔobcAB. (a) Growth of B. glumae strains in LB broth buffered to pH 7.0 with 70 mM Tris measured using a spectrophotometer. Equal numbers of cells (5 × 10⁷) were inoculated into 250‐mL culture flasks containing 40 mL of indicated growth medium and grown at 37°C with shaking. (b) At 6‐h intervals, a portion of the bacterial culture was aseptically removed to measure the medium pH. (c) The viable cell number of B. glumae, ΔdbcA, and ΔobcAB. Aliquots were taken at the indicated time points, serially diluted, and plated on LB agar plates containing 10 μg/mL nitrofurantoin. Colonies were counted after 48 h at 37°C. The data are presented as mean ± standard deviation (SD). Each experiment was repeated three times with three independent biological replicates.

FIGURE 2

Cation and colistin sensitivity of Burkholderia glumae ΔdbcA. (a) Minimum inhibitory concentration (MIC) of B. glumae wild type (336gr‐1) and ΔdbcA in LB broth for sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), magnesium sulphate (MgSO₄), manganese chloride (MnCl₂), ferric chloride (FeCl₃), and aluminium chloride (AlCl₃). The red arrows indicate the approximate MIC. (b) Divalent cation sensitivity on solid medium. Ten‐fold serially diluted cells of B. glumae and ΔdbcA transformed with control vector (vec) and pSC301 (dbcA) were spotted and grown on LB agar containing 100 μg/mL trimethoprim. For determination of cation sensitivity, plates were supplemented with either CaCl₂, MgSO₄, or MnCl₂ at the indicated concentrations. For determination of colistin sensitivity, plates were supplemented with either 0 or 100 μg/mL colistin. Sodium oxalate (Na₂C₂O₄) was added to plates at a concentration of 50 mM to test cation and colistin sensitivity in the presence of external oxalate. LB medium pH was set to 5.5 with hydrochloric acid to test the cation and colistin sensitivity in acidic pH. PC, positive control; NC, negative control. Each experiment was repeated three times with three independent biological replicates. Representative plates are shown.

FIGURE 3

Oxalic acid levels and acyl‐homoserine lactone (AHL) accumulation during growth of Burkholderia glumae wild type (336gr‐1), ΔdbcA, and ΔobcAB. (a) Oxalic acid production in LB broth buffered to 7.0 with 70 mM Tris. Inset bar graph shows oxalic acid levels at 6 h. Equal numbers of cells (5 × 10⁷) were inoculated into either unbuffered or buffered LB broth and grown at 37°C with shaking. Culture supernatants of B. glumae strains were collected by centrifugation at the indicated time points and the oxalic acid level was measured. (b) AHL quantification from culture supernatant of indicated strains grown in buffered LB broth based on β‐galactosidase activity. Representative wells are shown. N‐octanoyl homoserine (C8‐HSL, 10 μM) was added to the positive control, while no C8‐HSL was added to the negative control. The data are presented as mean ± standard deviation (SD). Each experiment was repeated three times with three independent biological replicates. Asterisks indicate a statistically significant difference between B. glumae and ΔdbcA. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 4

Expression of toxA, toxH, and obcA is down‐regulated in Burkholderia glumae ΔdbcA. (a) Relative normalized expression levels of toxA, toxH, and obcA in B. glumae wild type (336gr‐1) and ΔdbcA. (b) Relative normalized expression levels of qsmR, tofI, tofR, toxJ, and toxR in B. glumae and ΔdbcA. The data are presented as mean ± standard deviation (SD). Each experiment was repeated three times with three independent biological replicates. The statistical significance of differences between B. glumae wild type and ΔdbcA was calculated using the unpaired Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 5

Sodium bicarbonate (NaHCO₃) reduces oxalic acid production in Burkholderia glumae. (a, b) Growth and culture medium pH of B. glumae in buffered LB broth with or without 5 mM NaHCO₃. Equal numbers of cells (5 × 10⁷) were inoculated into culture flasks containing buffered LB medium supplemented with either 0 or 5 mM NaHCO₃. Bacterial cultures were grown at 37°C with shaking for 48 h. (c) Oxalic acid measurement of B. glumae grown in buffered LB broth with or without 5 mM NaHCO₃. (d) Acyl‐homoserine (AHL) quantification from culture supernatant of B. glumae grown in buffered LB broth with or without 5 mM NaHCO₃ based on β‐galactosidase activity. (e) Representative wells are shown. N‐octanoyl homoserine (C8‐HSL, 10 μM) was added to the positive control, while no C8‐HSL was added to the negative control. (f) Expression levels of toxA, toxH, and obcA in B. glumae grown in buffered LB broth with or without NaHCO₃. The data are presented as mean ± standard deviation (SD). Each experiment was repeated three times with three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 6

N‐octanoyl homoserine (C8‐HSL) restores oxalic acid production in Burkholderia glumae ΔdbcA. (a, b) Growth and culture medium pH of B. glumae wild type (336gr‐1) and ΔdbcA in buffered LB broth with or without 5 μM C8‐HSL. Equal numbers of cells (5 × 10⁷) were inoculated in a culture flask containing buffered LB broth and grown at 37°C with shaking. (c) Oxalic acid measurement of B. glumae and ΔdbcA grown in buffered LB broth with or without C8‐HSL. (d) Toxoflavin production by B. glumae and ΔdbcA grown in buffered LB broth with or without C8‐HSL. (e) Expression levels of toxA and toxH, and obcA in B. glumae and ΔdbcA grown in buffered LB broth with or without C8‐HSL. The data is presented as mean ± standard deviation (SD). Each experiment was repeated three times with three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 7

Inactivation of dbcA or treatment with NaHCO₃ results in a series of events leading to loss of toxoflavin production and virulence of Burkholderia glumae. Secretion of oxalic acid lowers the pH of the bacterial environment, which prevents nonenzymatic degradation of quorum sensing (QS) AHL signalling molecules (Yates et al., 2002). Plants respond to bacterial infection by producing metabolites that cause alkalinization of the apoplastic space (Geilfus, ; Nachin & Barras, ; O'Leary et al., 2016). QS activates expression of the tox operons required for virulence and the obc operon for oxalic acid synthesis (Kim et al., ; Nakata & He, 2010). Reduction of oxalic acid secretion by B. glumae ΔdbcA or exposure to NaHCO₃ prevents acidification, interfering with QS and tox expression, which in turn reduces the virulence of B. glumae (Goo et al., 2012). Loss of QS also represses the expression of the obc operon (Goo et al., 2017) and further reduces oxalic acid production and potentially amplifies the alkaline pH conditions.