Pyridine-2,6-Bis(Thiocarboxylic Acid) Produced by Pseudomonas stutzeri KC Reduces and Precipitates Selenium and Tellurium Oxyanions

Siderophores are iron-specific chelators that are produced and excreted by microorganisms under iron-limiting conditions as a part of an iron acquisition system. Some siderophores chelate metals other than iron, forming soluble or insoluble metal compounds and affecting the mobility and toxicity of those metals (8, 17, 47). Pyridine-2,6-bis(thiocarboxylic acid) (pdtc) is a siderophore produced by Pseudomonas stutzeri KC and P. putida DSM 3601 and DSM 3602 that has fortuitous carbon tetrachloride degradation activity (21, 30). Recent research has shown that pdtc promotes iron transport into the cell (25). P. stutzeri KC, its spontaneous pdtc-negative mutant CTN1, and other P. stutzeri strains also produce proferrioxamines (pFOs), which are probably their primary siderophores (A. Zawadzka et al., unpublished data). pdtc is a broad-range metal chelator; it chelates many transition metals, some heavy metals, lanthanides, and actinides. In general, micronutrient metals chelated by pdtc are soluble, while toxic metals form insoluble precipitates with pdtc (8, 39). Selenium and tellurium oxyanions are among the toxic metalloids precipitated by pdtc and pdtc-producing Pseudomonas stutzeri KC cultures. In nature selenium is present in igneous rocks and fossil fuels (19). The element is a metalloid that can exist in four oxidation states. Under aerobic conditions, selenium is present as toxic and soluble selenate (SeO42−) or selenite (SeO32−) or as insoluble and nontoxic elemental selenium (Se0). It is an essential micronutrient at low concentrations but is toxic at higher concentrations. Selenium is incorporated by organisms as selenide, and in high concentrations it substitutes for sulfur in cysteine and methionine, adversely affecting the integrity of proteins such as α-keratin and inhibiting the activity of enzymes such as glutathione peroxidase. Many bacteria have been shown to reduce selenite to Se0, thereby eliminating its toxicity (27, 34, 31); however, the mechanism of detoxification is unclear. Elemental selenium deposits were found in the cytoplasm (18, 19), in the periplasmic space (14), and outside the cells (48) of various organisms, suggesting that different mechanisms might be involved. Selenium can be reduced enzymatically by broad substrate-specific nitrate reductases (35), molybdenum-containing enzymes (4), or specific selenate reductases in an energy-conserving process (36). It has been suggested that the cellular pool of thiols, including glutathione (GSH), is involved in the reduction and resulting detoxification of both selenite and tellurite (18, 43). When all reduced GSH in a cell is converted to GS-Se-SG, the cell loses its principal reducing buffer and thus its nonspecific defense against selenium toxicity. Finally, sulfate-reducing bacterial (SRB) biofilms were found to precipitate elemental selenium and sulfur during sulfate-reducing growth as a nonspecific means of selenium removal (16). Tellurium is another highly toxic metalloid from group 16 of the periodic table. Tellurium is a relatively rare element found in nature in the form of metal tellurides (e.g., Pb, Cu, Ag, Au, and Sb). Industrial waste discharge sites can contain elevated concentrations of Te (2). Te(IV) as tellurite (TeO32−) is more toxic to most gram-negative bacteria than Se(IV) in the form of selenite. The MIC for bacteria lacking resistance determinants is in the order of 1 to 2 μg ml−1 for TeO32− (41) and 1 mg ml−1 for SeO32− (44). This is the reason tellurite was used as an antibacterial agent for the treatment of bacterial infections in the preantibiotic era and is still a component of some selective growth media (41). Bacterial resistance to tellurite is poorly understood but is thought to be associated with tellurite reduction and precipitation of metallic tellurium (27, 34, 38). It has been suggested that tellurite entering cells can be reduced by membrane-associated nitrate reductases (1, 35). Once inside the cell, glutathione and other thiol-carrying molecules are the main mediators of tellurite reduction (43, 45). Also, terminal oxidases of the respiratory chain in gram-negative bacteria are involved in the reduction of tellurite (42). Other mechanisms, including cysteine-metabolizing enzymes and methyl transferases, may be important resistance mechanisms against tellurite toxicity (3, 41). Finally, similarly to what occurs with selenite, tellurite reduction and precipitation by sulfate-reducing bacteria has been reported (27). Our current research investigated the nature of selenium and tellurium precipitates formed upon interaction with pdtc and the role of pdtc in detoxification of the bacterial environment. Here we propose a chemical mechanism for interactions of pdtc with selenite and tellurite. We identified the initial precipitates of Se and Te as insoluble selenides and tellurides of pdtc and the hydrolysis products of pdtc. Our data suggest that hydrolysis and oxidation of those initial precipitates leads to the formation of elemental Se and Te. We compared the reduction and precipitation products of selenite and tellurite mediated by synthetic, high-purity pdtc to the reaction products formed by pdtc-producing P. stutzeri KC and the pdtc-negative mutant strain CTN1. The nature of the precipitates formed in bacterial cultures of P. stutzeri KC was similar to the in vitro reaction products formed by synthetic pdtc. Electron microscopy analyses showed that in strain KC, elemental selenium and tellurium accumulated mainly extracellularly; however, pdtc did not completely prevent the accumulation of intracellular Se0 and Te0 under our experimental conditions. The use of pdtc by P. stutzeri KC to reduce selenite and tellurite is to our knowledge a unique system utilizing a siderophore to carry thiols to the extracellular environment that we describe here for the first time in bacteria. Our results add a novel function to the known activities of pdtc that is linked to detoxification of the bacterial habitat (5).