Impact of Silver(I) on the Metabolism of Shewanella oneidensis

Anaerobic cultures of Shewanella oneidensis MR-1 reduced toxic Ag(I), forming nanoparticles of elemental Ag(0), as confirmed by X-ray diffraction analyses. The addition of 1 to 50 μM Ag(I) had a limited impact on growth, while 100 μM Ag(I) reduced both the doubling time and cell yields. At this higher Ag(I) concentration transmission electron microscopy showed the accumulation of elemental silver particles within the cell, while at lower concentrations the metal was exclusively reduced and precipitated outside the cell wall. Whole organism metabolite fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells grown in a range of silver concentrations, confirmed that there were significant physiological changes at 100 μM silver. Principal component-discriminant function analysis scores and loading plots highlighted changes in certain functional groups, notably, lipids, amides I and II, and nucleic acids, as being discriminatory. Molecular analyses confirmed a dramatic drop in cellular yields of both the phospholipid fatty acids and their precursor molecules at high concentrations of silver, suggesting that the structural integrity of the cellular membrane was compromised at high silver concentrations, which was a result of intracellular accumulation of the toxic metal.Silver is an element that has been used widely in industrial processes as diverse as photographic processing, catalysis, mirror production, electroplating, alkaline battery production, and jewelry making (18). It has been known for some time that silver ions and silver-based compounds can be highly toxic to microorganisms, and with increasing concern about pathogenic “superbugs” with high resistance to conventional antibiotics, silver is attracting much interest as a potential biocide (11, 18, 36, 42). Silver has no known physiological functions and can exist in several oxidation states, although it is most commonly encountered in its elemental [Ag(0)] and monovalent [Ag(I)] forms. Although use of nanoscale elemental Ag(0) as a biocide has been increasing, for example, in wound dressings and as an antimicrobial coating on consumer products, little is known about its mode of toxicity. This is despite the surprising ability of actively growing Fe(III)-reducing bacteria such as Geobacter sulfurreducens to precipitate nanoscale Ag(0) particles within and around the cell surface via reduction of Ag(I) (18). Ionic Ag(I), in contrast, has been the focus of more studies on the mode of metal toxicity. Previous research showed that silver ions have antimicrobial activities against a wide diversity of bacteria (19). They have been shown to disrupt the respiratory chain of Escherichia coli (3) and inhibit the exchange of phosphate and its uptake (34). Ag(I) has also been linked to copper metabolism in E. coli, potentially competing with copper binding sites on the cell surface and subsequent copper transport into the cell (8). However, the toxicity of silver is not limited to prokaryotes, as long-term exposure in humans can cause argyria, impaired night vision, and abdominal pain (31, 32, 36). The detailed mechanism of toxicity in prokaryotes or eukaryotes remains to be identified, although it has been proposed that silver ions react with cellular proteins via SH groups (16), leading to the disruption of cellular metabolism.Microbial cells have evolved an extremely diverse range of mechanisms to survive high concentrations of toxic metals. The mechanisms invoked include biosorption, bioaccumulation, special efflux systems, alteration of solubility and toxicity via reduction or oxidation, extracellular complexation or precipitation of metals, and lack of specific metal transport systems (1). For example, for silver ions the bacterial cell wall can be an efficient permeability barrier to block the uptake of metal (21), with additional complexation in the periplasm by specific silver-binding proteins (35). Redox transformations also offer the potential to detoxify Ag(I) ions, e.g., through the reduction to insoluble elemental Ag(0) (30). In addition, the energy-dependent efflux of toxic Ag(I) is perhaps the best-studied resistance mechanism for silver, mediated via ATPases and chemiosmotic cation/protons antiporters (9).Shewanella spp., Gram-negative, dissimilatory metal-reducing bacteria, can use a wide variety of terminal electron acceptors for growth (23, 39), including high oxidation state metals such as Fe(III), Mn(IV), Cr(VI), U(VI), and Au(III) (5, 17, 26, 28, 41). Shewanella species also have the potential to reduce Ag(I), given their similar activities against Au(III), and the reduction of Ag(I) to form nanoscale deposits of Ag(0) within the cell has been documented for other Fe(III)-reducing bacteria (18). This metabolic versatility offers considerable potential for bioremediation applications, for example, via reduction of U(VI) to insoluble U(IV) (5, 17, 26, 28, 41), and the recovery of precious metals such as silver and gold via reductive precipitation. It also offers an interesting model organism to study the metabolism of toxic metals such as silver, including the physiological impact of ionic Ag(I) and nanoscale Ag(0).This paper describes interactions of Shewanella oneidensis MR-1 with various concentrations of Ag(I), including demonstrations of the reduction and deposition of silver nanoparticles under anaerobic conditions. A range of techniques, including X-ray diffraction (XRD) and analytical transmission electron microscopy (TEM), were used to investigate the nature and cellular localization of the precipitates, while Fourier transform infrared (FT-IR) spectroscopy metabolic profiling techniques were used to identify the impact of toxic metal accumulation on the cell. The disruption of membrane integrity was implied by these investigations and confirmed by fatty acid methyl ester (FAME) analysis, which showed a dramatic decrease in the quantities of membrane lipid components.