Cobalamin Protection against Oxidative Stress in the Acidophilic Iron-oxidizing Bacterium Leptospirillum Group II CF-1

Abstract

Members of the genus Leptospirillum are aerobic iron-oxidizing bacteria belonging to the phylum Nitrospira. They are important members of microbial communities that catalyze the biomining of sulfidic ores, thereby solubilizing metal ions. These microorganisms live under extremely acidic and metal-loaded environments and thus must tolerate high concentrations of reactive oxygen species (ROS). Cobalamin (vitamin B12) is a cobalt-containing tetrapyrrole cofactor involved in intramolecular rearrangement reactions and has recently been suggested to be an intracellular antioxidant. In this work, we investigated the effect of the exogenous addition of cobalamin on oxidative stress parameters in Leptospirillum group II strain CF-1. Our results revealed that the external supplementation of cobalamin reduces the levels of intracellular ROSs and the damage to biomolecules, and also stimulates the growth and survival of cells exposed to oxidative stress exerted by ferric ion, hydrogen peroxide, chromate and diamide. Furthermore, exposure of strain CF-1 to oxidative stress elicitors resulted in the transcriptional activation of the cbiA gene encoding CbiA of the cobalamin biosynthetic pathway. Altogether, these data suggest that cobalamin plays an important role in redox protection of Leptospirillum strain CF-1, supporting survival of this microorganism under extremely oxidative environmental conditions. Understanding the mechanisms underlying the protective effect of cobalamin against oxidative stress may help to develop strategies to make biomining processes more effective.

Keywords: Leptospirillum group II CF-1; bioleaching; cobalamin; heavy metals; oxidative stress; vitamin B12.

FIGURE 1

Effect of cobalamin on ROS generation. Cytoplasmic ROS content was estimated by measuring activation of the fluorescent-probe H₂DCFDA in Leptospirillum CF-1 cells treated with oxidative agents as indicated under Section “Materials and Methods.” Fluorescence values were expressed as relative fluorescence units (RFU). Bars represent the average of three independent experiments ± standard deviations. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 2

Effect of cobalamin on lipid peroxidation. Lipid peroxidation products, expressed as malondialdehyde (MDA) equivalents, were determined as thiobarbituric acid-reactive substances present in crude extracts in Leptospirillum CF-1 cells as indicated under Section “Materials and Methods.” Bars represent the average of three independent experiments ± standard deviations. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 3

Effect of cobalamin on thioredoxin and thioredoxin reductase activity. To measure Trx activity (A), the reduction of the alpha-chain of insulin was monitored at 650 nm as described under Section “Materials and Methods.” TR activity (B) was monitored by following the reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) at 412 nm. The activity in the control reaction corresponds to 100%. Data represent the average of two independent experiments (lines on top of bars indicate value ranges). ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 4

Effect of cobalamin on CcP activity. The activity was followed spectrophotometrically at 550 nm as indicated under Section “Materials and Methods.” Data represent the average of two independent experiments (lines on top of bars indicate value ranges). One unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of ferrocytochrome c per min. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 5

Effect of cobalamin on superoxide dismutase activity. The activity was measured by following the inhibition of NBT-reduction at 560 nm as described under Section “Materials and Methods.” Data represent the average of two independent experiments (lines on top of bars indicate value ranges). One unit (U) is defined as the amount of enzyme necessary to decrease the reference rate to 50% of maximum inhibition. ^∗∗P < 0.01.

FIGURE 6

Relative mRNA levels of the cbiA gene in Leptospirillum CF-1. Bacteria were treated with 260 Fe₂(SO₄)₃ for 30 min. Data were normalized by the 16S rRNA. Data represent the average of two independent experiments (bar indicates the value range). ^∗P < 0.05.