Osmotic Imbalance, Cytoplasm Acidification and Oxidative Stress Induction Support the High Toxicity of Chloride in Acidophilic Bacteria

Abstract

In acidophilic microorganisms, anions like chloride have higher toxicity than their neutrophilic counterparts. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm. We predicted that intracellular acidification produces an increase in respiratory rate and generation of reactive oxygen species, and so oxidative stress can also be induced. In this study, the multifactorial effect as inducing osmotic imbalance, cytoplasm acidification and oxidative stress in the iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 exposed to up to 150 mM NaCl was investigated. Results showed that chloride stress up-regulated genes for synthesis of potassium transporters (kdpC and kdpD), and biosynthesis of the compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displayed a transcriptionally regulated response against oxidative stress induced by chloride. Altogether, these data reveal that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. Exploration of the adaptive mechanisms to anion stress in iron-oxidizing acidophilic microorganisms may result in new strategies that facilitate the bioleaching of ores for recovery of precious metals in presence of chloride.

Keywords: Leptospirillum spp.; acidophiles; bioleaching; chloride; compatible solutes; osmolarity; oxidative stress.

FIGURE 1

Relative mRNA levels of osmotic stress related genes in L. ferriphilum DSM 14647. Relative expression of K⁺ transporter genes was evaluated (A) in cells treated with 100 mM NaCl for 5, 20, and 50 min. Relative expression of genes for biosynthesis of (hydroxy)ectoine (B) and trehalose (C) was evaluated in cells treated with 100 mM NaCl for 0 and 80 min. Data were normalized by the 16S rRNA. Data represent the average of three independent experiments (bars indicate the value range). Statistical analysis was carried out by ANOVA and t-Test.

FIGURE 2

Effect of NaCl exposure on intracellular content of compatible solutes in L. ferriphilum DSM 14647. Content of ectoine and hydroxyectoine (A), and trehalose (B) in cells exposed to 100 mM NaCl for 90 min. The data represent the average of 3 independent experiments (bars indicate the value range). Statistical analysis was carried out by ANOVA and t-Test. N.D.: not detected.

FIGURE 3

Effect of NaCl exposure on intracellular pH (pH_in) in L. ferriphilum DSM 14647. Effect of NaCl concentration on pH_in of cells exposed for 60 min to the stress condition (A). Effect of the exposure time in cells exposed to 100 mM NaCl (B). The data represent the average of 3 independent experiments (bars indicate the value range). Statistical analysis was carried out by ANOVA Test.

FIGURE 4

Effect of NaCl concentration on oxygen consumption rate in L. ferriphilum DSM 14647. The data represent the average of three independent experiments (bars indicate the value range). Statistical analysis was carried out by ANOVA Test.

FIGURE 5

ROS generation in L. ferriphilum DSM 14647. Effect of NaCl concentration (A) and exposure time (B) on intracellular ROS generation in the bacteria. The concentration effect was evaluated in cells exposed to NaCl for 60 min. The effect of exposure time was evaluated in cells exposed to 100 mM NaCl. Cytoplasmic ROS content was expressed as relative fluorescence units (RFU) of the activated fluorescent probe H₂DCFDA per mg of protein.

FIGURE 6

Antioxidant protein activity in L. ferriphilum DSM 14647. The cytochrome c peroxidase (A) and thioredoxin activity (B) in cells exposed to 100 mM NaCl. The data represent the average of three independent experiments (bar indicates the value range). Statistical analysis was carried out by ANOVA Test.

FIGURE 7

Relative mRNA levels of oxidative stress related genes in L. ferriphilum DSM 14647. Relative expression of cytochrome c peroxidase (ccp) and the thioredoxin (trx) encoding genes was evaluated in cells treated with 100 mM NaCl. Data were normalized by the rrsB gene. Data represent the average of three independent experiments (bars indicate the value range). Statistical analysis was carried out by ANOVA Test.

FIGURE 8

Multifaceted effect of NaCl in L. ferriphilum DSM 14647.