Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect

Abstract

Fluoride ion (F(-)) is a ubiquitous environmental threat to microorganisms, which have evolved a family of highly selective "Fluc" F(-) channels that export this inhibitory anion from their cytoplasm. It is unclear, however, how a thermodynamically passive mechanism like an ion channel can protect against high concentrations of external F(-). We monitored external F(-) concentrations in Escherichia coli suspensions and showed that, in bacteria lacking Fluc, F(-) accumulates when the external medium is acidified, as a predicted function of the transmembrane pH gradient. This weak acid accumulation effect, which results from the high pKa (3.4) and membrane permeability of HF, is abolished by Fluc channels. We also found that, although bacterial growth is inhibited by high concentrations of F(-), bacteria can withstand cytoplasmic F(-) at levels a hundred times higher than those that inhibit proliferation, resuming growth when the F(-) challenge is removed.

Figure 1.

Weak acid accumulation effect for F⁻. A cell devoid of any membrane pathway for F⁻ anion is depicted in a medium of pH 5.0. If cytoplasmic pH is regulated near neutrality, under such conditions F⁻ will necessarily accumulate in the cell because of the high pKa and membrane permeability of HF.

Figure 2.

F⁻ inhibition of E. coli growth at varying external pH. Growth curves are shown for wild-type and ΔFluc E. coli in media adjusted to the indicated pH and NaF concentrations.

Figure 3.

F⁻ uptake by E. coli. (A) Raw data traces of extracellular F⁻ concentration in suspensions of wild-type and ΔFluc E. coli in response to acid exposure or control buffer without cells. Calibration pulse of 0.1 mM NaF (closed triangle) and addition of HCl to drop the pH from 7.5 to 5.5 (open triangle) are indicated. (B) Representative F⁻ electrode traces of F⁻ uptake by ΔFluc E. coli cells in 0.5 mM F⁻ medium, with changing external pH to the indicated values at t = 0. (C) F⁻ uptake at pH 5.3 in the indicated concentrations of external F⁻.

Figure 4.

F⁻ uptake by ΔFluc E. coli cells as a function of pH or F⁻ concentration. (A) Ratio of internal and external [F⁻] with varying pH. The dashed line has a slope of 1, as predicted by Eq. 1. Inset plots data directly against medium pH. (B) Ratio of internal and external F⁻. The solid line is a fit to the data with slope of 54, corresponding to a transmembrane pH differential of 1.7 units. F⁻ uptake was determined as in Fig 3. Each point shows mean (±SEM) of three independent measurements. Internal pH was assumed to remain constant at 7.0.

Figure 5.

F⁻ exposure during bacterial growth. ΔFluc E. coli in log-phase growth was treated with 0.5 mM NaF at the indicated pH. After 2 h, the F⁻ was washed away, the pH was returned to 7.0, and optical density of the culture was monitored. Dashed curve is control without F⁻ exposure.

Figure 6.

Bacterial survival after F⁻ exposure. Wild-type or ΔFluc E. coli culture was incubated with 0.5 mM NaF at the indicated pH for the indicated time. Afterward, cells were plated on neutral-pH LB plates, and colonies were counted after 16 h. The number of colonies was normalized against a wild-type culture that was not treated with F⁻ (dashed line indicates 1.4 × 10⁹ CFU/ml). Points represent means ± SEM of three independent measurements.