ABSTRACT
Silicon (Si) is considered to be a "quasiessential" element for most living organisms. However, silicate uptake in bacteria and its physiological functions have remained obscure. We observed that Si is deposited in a spore coat layer of nanometer-sized particles in Bacillus cereus and that the Si layer enhances acid resistance. The novel acid resistance of the spore mediated by Si encapsulation was also observed in other Bacillus strains, representing a general adaptation enhancing survival under acidic conditions.
Subject(s)
Acids/pharmacology , Bacillus cereus/drug effects , Bacillus cereus/metabolism , Silicon/metabolism , Silicon/physiology , Spores, Bacterial/drug effects , Spores, Bacterial/metabolism , Bacillus cereus/genetics , Bacillus cereus/radiation effects , Bacillus cereus/ultrastructure , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/radiation effects , Hydrochloric Acid/pharmacology , Hydrofluoric Acid/pharmacology , Microscopy, Electron, Scanning , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sodium Hydroxide/pharmacology , Spores, Bacterial/genetics , Spores, Bacterial/radiation effects , Spores, Bacterial/ultrastructure , Temperature , Ultraviolet Rays/adverse effectsABSTRACT
We previously reported a silica-binding protein, designated Si-tag, which can be used to immobilize proteins on silica surfaces. Here, we constructed a fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A for oriented immobilization of antibodies on a silicon wafer whose surface is oxidized to silicon dioxide (silica). The fusion protein, Si-tagged protein A, strongly bound to the silica surface with a dissociation constant of 0.31 nM. Time-of-flight secondary ion mass spectrometry analysis of the silicon wafer coated with Si-tagged protein A, combined with principal component analysis and mutual information, demonstrated that protein A is localized on the outermost surface of the bound protein layer. Immunoglobulin G (IgG) was immobilized both on the silicon wafer coated with Si-tagged protein A and, as a control, directly on the intact silicon wafer via physical adsorption. The silicon wafer coated with Si-tagged protein A bound 30-70% more IgG than the uncoated silicon wafer, whereas the antigen-binding activity was 4- to 5-fold higher for the former, indicating that IgG was functionally immobilized on the silicon wafer via Si-tagged protein A in an oriented manner.
Subject(s)
Antibodies, Immobilized/chemistry , Immunoglobulin G/chemistry , Silicon Dioxide/chemistry , Staphylococcal Protein A/chemistry , Antigen-Antibody Reactions , Binding Sites , Recombinant Fusion Proteins/chemistry , Spectrometry, Mass, Secondary Ion , Staphylococcus aureus/chemistry , Surface PropertiesABSTRACT
Targeting functional proteins to specific sites on a silicon device is essential for the development of new biosensors and supramolecular assemblies. Using intracellular lysates of several bacterial strains, we found that ribosomal protein L2 binds tightly to silicon particles, which have surfaces that are oxidized to silica. A fusion of E. coli L2 and green fluorescence protein adsorbed to the silica particles with a K(d) of 0.7 nM at pH 7.5 and also adsorbed to glass slides. This fusion protein was retained on the glass slide even after washing for 24 h with a buffer containing 1 M NaCl. We mapped the silica-binding domains of E. coli L2 to amino acids 1-60 and 203-273. These two regions seemed to cooperatively mediate the strong silica-binding characteristics of L2. A fusion of L2 and firefly luciferase also adsorbed on the glass slide. This L2 silica-binding tag, which we call the "Si-tag," can be used for one-step targeting of functional proteins on silica surfaces.
