Stimulation of Expression of a Silica-Induced Protein (Sip) in Thermus thermophilus by Supersaturated Silicic Acid

The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75°C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO₂), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe³⁺ ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_006461","term_id":"55979969","term_text":"NC_006461"}}NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe³⁺-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_005835","term_id":"46198308","term_text":"NC_005835"}}NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.Occurring mainly in the form of silica (SiO₂), silicon (Si) is the second-most abundant element in the earth''s crust, accounting for 28.8% of the earth''s mass (34). SiO₂ exists as monosilicic acid (Si(OH)₄) in aqueous solution, as represented in the following equation: SiO₂ + 2H₂O · (Si(OH)₄). The solubility of silica greatly depends on temperature, pH (17), and salt concentration, among other parameters (27). As the temperature of a silicic acid solution declines, its concentration can exceed the solubility of amorphous silica. Under those conditions, silicic acid polymerizes to form polysilisic acid, which is relatively stable in aqueous solution because the repulsion between the negative charges on its surface keeps it from readily aggregating and precipitating. In a geothermal reservoir, at high temperature and pressure, the silicic acid concentration at equilibrium shows the solubility of quartz. However, when that geothermal water is discharged to the surface, the silicic acid concentration becomes supersaturated as the water boils, frequently leading to the formation of siliceous deposits called “silica sinter” (11). Microscopic observation of such siliceous deposits reveals many microbe-like structures (20), and it has been suggested that these fossils represent archean microorganisms that grew in the hot, supersaturated fluids (26). There have been a number of experimental studies carried out with the aim of characterizing the physical changes associated with various bacteria during silicification (23, 26, 31, 33, 37); however, the effect of silica on the bacterial habitat in geothermal environments and the mechanism by which siliceous deposits are formed remain unexplained.Recent studies have shown that biosilicification in geothermal areas reflects the activities of various thermophilic microorganisms (15, 23, 29). For instance, geothermal water and the water discharged from hydrothermal vents contain a high concentration of silicic acid, and biogenic textures covered with amorphous silica have been found in areas around both sources (12, 22). Moreover, in a study of experimental silicification, interactions were observed between silica and Sulfurihydrogenibium azorense, a representative member of the order Aquificales (26). Still, the effect of silica on the bacterial habitat in thermal environments and the molecular properties affecting aggregation and siliceous deposition remain poorly understood.Our previous studies have focused on the effect of bacteria on the formation of siliceous deposits in geothermal water (18, 21). Siliceous deposits (called silica scale) that form in pipelines and on surface equipment in geothermal power plants cause serious economic problems related to energy loss and to plant maintenance throughout the world (38). We also observed that a Thermus strain isolated from silica scale (TMY) was able to efficiently generate and deposit amorphous silica in vitro, beginning in the latter part of the log growth phase (19). On the basis of its morphological, physiological, and genetic properties, the organism was identified as a strain of Thermus thermophilus, and its distinct properties were indicative of the microdiversity of T. thermophilus strains (14). Thermus strains are the predominant heterotrophs in natural geothermal and hydrothermal habitats, and thus far, the genomes of T. thermophilus strains HB8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AP008226","term_id":"55771382","term_text":"AP008226"}}AP008226) and HB27 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE017221","term_id":"46197919","term_text":"AE017221"}}AE017221) have been sequenced completely (16). Given its genomic similarity to T. thermophilus HB8, we analyzed the genetic information of strain TMY in that context.Here we report the effect of silicic acid concentration on the growth of T. thermophilus TMY, which was isolated from a siliceous deposit formed at a geothermal electric power plant. Notably, supersaturated silicic acid markedly stimulated expression of one cell envelope protein, which we named silica-induced protein (Sip). Induction of Sip expression occurred rapidly after the cells were exposed to supersaturated silicic acid, and the amino acid sequence of Sip showed significant similarity with the Fe³⁺ ABC transporters observed in other Thermus strains. These results shed new light on the growth and biosilisification associated with thermophiles in geothermal environments.