Bactericidal actions of the silver ion on Escherichia coli as a model microorganism were studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). EFTEM observations demonstrated that the silver ion readily infiltrates the interior of E. coli, contrary to the early hypothesis that it resides initially in the cell membrane area. Furthermore, 2-DE and MALDI-TOF MS indicated that the expression of a ribosomal subunit protein as well as that of some other enzymes and proteins is affected by the silver ion. The present results demonstrate for the first time that one of the major bactericidal functions of the silver ion is its interaction with the ribosome and the ensuing inhibition in expression of the enzymes and proteins essential to ATP production.The silver ion is known to have antimicrobial effects, and it is increasingly utilized in electric appliances, such as washing machines. Many papers have been published on its effects, and early authors hypothesized that the silver ion primarily affects the function of membrane-bound enzymes, such as those in the respiratory chain, through binding to thiol groups (1, 4, 7). However, proteomic insight into the detailed mechanism is still lacking. In this paper, bactericidal actions of a silver ion solution on Escherichia coli as a model microorganism was studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).Experimental details are as follows. An aqueous solution with a silver ion concentration of 900 ppb was electrolytically prepared by applying a current of 12.5 mA for 28 s between two silver plate electrodes installed in water. The residual chlorine content in water was less than 0.2 mg/liter. The silver ion concentration in the obtained solution was measured with a Hitachi Z-5010 polarized Zeeman atomic absorption spectrophotometer. As a reference, a 0-ppb solution was also prepared, without electrolysis.As a model microorganism, gram-negative E. coli NBRC-3972 was used. Ten milliliters of the aqueous suspension with 10 7 to ϳ10 8 CFU/ml E. coli reacted with 90 ml of a silver ion solution for 30 min, 3 h, and 24 h. Viable cells were enumerated on an agar plate containing 1 g/liter glucose, 2.5 g/liter yeast extract, and 5 g/liter tryptone by the colony count method, after incubation at 35°C for 24 h.For morphological observation and elemental analysis using EFTEM, E. coli cells that reacted with the silver ion were first fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C. After being washed with 0.1 M cacodylate buffer, the suspension was osmicated in 2% osmium tetroxide at 4°C for 4 h and then dehydrated repeatedly with increased concentrations of ethanol. The fixed cells were embedded in epoxy resin at 60°C for 48 h and sliced into an approximately...