Silica-Induced Protein (Sip) in Thermophilic Bacterium Thermus thermophilus Responds to Low Iron Availability

Abstract

Thermus thermophilus HB8 expresses silica-induced protein (Sip) when cultured in medium containing supersaturated silicic acids. Using genomic information, Sip was identified as a Fe 3؉ -binding ABC transporter. Detection of a 1-kb hybridized band in Northern analysis revealed that sip transcription is monocistronic and that sip has its own terminator and promoter. The sequence of the sip promoter showed homology with that of the A -dependent promoter, which is known as a housekeeping promoter in HB8. Considering that sip is transcribed when supersaturated silicic acids are added, the existence of a repressor is presumed. DNA microarray analysis suggested that supersaturated silicic acids and iron deficiency affect Thermus cells similarly, and enhanced sip transcription was detected under both conditions. This suggested that sip transcription was initiated by iron deficiency and that the ferric uptake regulator (Fur) controlled the transcription. Three Fur gene homologues (TTHA0255, TTHA0344, and TTHA1292) have been annotated in the HB8 genome, and electrophoretic mobility shift assays revealed that the TTHA0344 product interacts with the sip promoter region. In medium containing supersaturated silicic acids, free Fe 3؉ levels were decreased due to Fe 3؉ immobilization on colloidal silica. This suggests that, because Fe 3؉ ions are captured by colloidal silica in geothermal water, Thermus cells are continuously exposed to the risk of iron deficiency. Considering that Sip is involved in iron acquisition, Sip production may be a strategy to survive under conditions of low iron availability in geothermal water. IMPORTANCEThe thermophilic bacterium Thermus thermophilus HB8 produces silica-induced protein (Sip) in the presence of supersaturated silicic acids. Sip has homology with iron-binding ABC transporter; however, the mechanism by which Sip expression is induced by silicic acids remains unexplained. We demonstrate that Sip captures iron and its transcription is regulated by the repressor ferric uptake regulator (Fur). This implies that Sip is expressed with iron deficiency. In addition, it is suggested that negatively charged colloidal silica in supersaturated solution absorbs Fe 3؉ ions and decreases iron availability. Considering that geothermal water contains ample silicic acids, it is suggested that thermophilic bacteria are always facing iron starvation. Sip production may be a strategy for surviving under conditions of low iron availability in geothermal water. S ilica (SiO 2 ) is one of the most abundant substances in the Earth's crust. In aqueous solution, silica dissolves as monosilicic acid, Si(OH) 4 , the solubility of which depends on conditions such as temperature, pH, and salt concentrations (1). In geothermal water at high temperature and pressure underground, silicic acids are in equilibrium with the quartz in rock layers, with concentrations reaching around 600 ppm at 300°C. When this geothermal water is discharged to the surface, where the solubility of amorphous silica is about 300 ppm...

Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

“…Although the maximum activity value achieved in a medium exchange culture was 3.6-fold higher than that observed before induction, this maximum activity was less than one-third of the value observed after an uninterrupted culture period in 10 mM silica. In a recent report, we similarly observed that medium exchange was ineffective in an E. coli-based protein expression system that utilizes silica as an inducer in advance [12]. As discussed in previous reports, this decrease in protein expression may be attributable to intracellular iron storage proteins.…”

“…We previously reported that Thermus strains produced a specific Sip in the presence of supersaturated silica [12]. This Sip exhibited high homology with a Fe 3+ -binding ABC transporter that plays a role in Fe 3+ uptake.…”

“…We previously reported the expression of a specific silica-induced protein (Sip) in T. thermophilus exposed to supersaturated silica [11]. We further identified this Sip as a Fe 3+ -binding ABC transporter encoded by a gene for which the transcription was regulated strictly by a ferric uptake regulator (Fur) via a previously reported mechanism [12]. Importantly, the sip promoter might be sufficiently strong to mediate heterologous expression, as indicated by the greater than tenfold increase in Sip expression under induced conditions relative to un-induced conditions and the prominent protein band of 35 kDa detected by Coomassie brilliant blue staining.…”

“…Despite those earlier findings, we selected wild-type HB27 as the host strain in our β-galactosidase assay because a previously reported qRT-PCR analysis revealed strong sip promoter activity (16-fold increase in response to 10 mM silica) [12]. As expected, exposure to 10 mM silica induced a sufficiently high level of β-galactosidase activity to enable a comparison between the induced and non-induced conditions.…”

“…The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using EcoRI and Nhe I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein (sip) promoter region [12] were cloned into pYK596 using an In-Fusion cloning kit. The Xho I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1.…”