Abstract
Antimonial compounds can be found as a toxic contaminant in the environment. Knowledge on mechanisms of microbial Sb oxidation and its role in microbial tolerance are limited. Previously, we found that Comamonas testosteroni S44 was resistant to multiple heavy metals and was able to oxidize the toxic antimonite [Sb(III)] to the much less toxic antimonate [Sb(V)]. In this study, transposon mutagenesis was performed in C. testosteroni S44 to isolate genes responsible for Sb(III) resistance and oxidation. An insertion mutation into iscR, which regulates genes involved in the biosynthesis of Fe-S clusters, generated a strain called iscR-280. This mutant strain was complemented with a plasmid carrying iscR to generate strain iscR-280C. Compared to the wild type S44 and iscR-280C, strain iscR-280 showed lower resistance to Sb(III) and a lower Sb(III) oxidation rate. Strain iscR-280 also showed lower resistance to As(III), Cd(II), Cu(II), and H2O2. In addition, intracellular γ-glutamylcysteine ligase (γ-GCL) activity and glutathione (GSH) content were decreased in the mutated strain iscR-280. Real-time RT-PCR and lacZ fusion expression assay indicated that transcription of iscR and iscS was induced by Sb(III). Results of electrophoretic mobility shift assay (EMSA) and bacterial one-hybrid (B1H) system demonstrated a positive interaction between IscR and its promoter region. The diverse defective phenotypes and various expression patterns suggest a role for IscR in contributing to multi-metal(loid)s resistance and Sb(III) oxidation via Fe-S cluster biogenesis and oxidative stress protection. Bacterial Sb(III) oxidation is a detoxification reaction.
Highlights
Antimony (Sb) belongs to subgroup 15 of the periodic table along with nitrogen (N), phosphorus (P), arsenic (As), and bismuth (Bi)
Four mutants have an insertion within iscR and two mutant have an insertion between iscR and iscS
The results demonstrated that the γ -glutamylcysteine ligase (γ -GCL) activity of iscR-280 was much lower than that of S44 and iscR-280C (Figure 5E), and the GSH content was positively correlated with γ -GCL activity (Figure 5F)
Summary
Antimony (Sb) belongs to subgroup 15 of the periodic table along with nitrogen (N), phosphorus (P), arsenic (As), and bismuth (Bi). The most common oxidation states found in nature are Sb(III) and Sb(V) (Li et al, 2013). Sb and its compounds are considered as pollutants by the Environmental Protection Agency of the United States (USEPA) and the European Union Molecular mechanisms of Sb resistance were mostly studied in the protozoan parasite Leishmania. In vitro studies suggested that genes encoding AQP1, PGPA, TDR1, and ACR2 in parasites are required for Sb(III) resistance (Decuypere et al, 2012). The best-known mechanism of resistance to Sb involves the detoxification of Sb(III) via conjugation to trypanothione [T(SH)2], which is a thiol compound in the protozoan parasite (Legare et al, 2001)
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