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 σ(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.

Importance: The 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.

FIG 1

(A) Genetic arrangement of the T. thermophilus HB8 sip locus. The position of the putative sip terminator sequence is illustrated. The locus tags of the genes are indicated in the arrows. (B) Northern blot analysis of sip transcription. Lane M, DIG-labeled RNA molecular markers; lane -Si, total RNA from cells cultivated in TM; lane +Si, total RNA from cells cultivated in TM containing 600 ppm silicic acids; lane -Fe, total RNA from cells cultivated in iron-deficient TM. Under each lane are the 16S rRNA bands before blotting of the ethidium bromide-stained gel. (C) Semiquantitative RT-PCR of TTHA1628 to TTHA1630. Lanes RT-1 to RT-5 correspond to the target regions shown in panel A. The PCR products were amplified with cDNA from cells cultivated in Si-containing medium (+Si) or normal medium (-Si). (D) Primer extension analysis of sip. Filled circle, the transcription initiation site (TIS) in the sequence; arrow, the TIS on the blot. A potential Shine-Dalgarno (SD) site is also shown. Putative −35 and −10 regions of the σ^A-like promoter are indicated. The deduced amino acids are shown below their respective codons.

FIG 2

(A) Relative levels of the sip transcript. Relative transcription levels were determined using qRT-PCR with RNA isolated from cells cultivated in TM containing 0, 200, 400, or 600 ppm silicic acids or iron-deficient TM. White numbers in bars, fold changes, compared to TM without silicic acids. The values are expressed as normalized intensity ± standard deviation (n = 3). P values were calculated using t tests. N.S., nonsignificant. (B) Fe³⁺ concentrations in the media used in this study. Black bars, total Fe³⁺ concentrations; white bars, free Fe³⁺ concentrations. The values are expressed as mean ± standard deviation (n = 3). *, P of <0.01.

FIG 3

Titration of apo-Sip with Fe³⁺. (A) UV-visible spectra of apo-Sip before and after the addition of Fe(NTA)₂ (0, 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mol eq). (B) Titration curve for the binding of Fe³⁺ to apo-Sip, based on the intensity of the LCMT band at 450 nm.

FIG 4

(A) Sequence alignment of Fur and Fur-like proteins from T. thermophilus HB8. The amino acid sequences of the Fur proteins from Escherichia coli strain K-12 substrain MG1655 (EcFur), Bacillus subtilis strain 168 (BsFur), and Helicobacter pylori strain 26695 (HpFur) and the Fur-like proteins from T. thermophilus HB8 (TTHA0255, TTHA0344, and TTHA1292) were aligned using ClustalW. Filled triangles, residues involved in metal-binding site 1 (S1); filled circles, residues involved in S2; open circles, residues involved in S3. Black shading, positions with 100 to 80% amino acid conservation; gray shading, positions showing 80 to 50% conservation. (B) Binding of Fur homologues to the 5′ region of sip. All lanes contained 2 nM end-labeled sip promoter DNA (Psip). Lanes 1 to 6, 0, 10, 50, 100, 500, and 1,000 nM TTHA0344 product, respectively; lane 7, same as lane 6 but with a 200-fold excess of unlabeled Psip DNA; lane 8, 1,000 nM TTHA0255 product; lane 9, 1,000 nM TTHA1292 product. Arrow C, Fur-DNA complex; arrow F, free DNA.

FIG 5

Schematic diagram of Fur-mediated regulation of sip induction by supersaturated silicic acids. Colloidal silica particles are generated under supersaturated conditions, and the negatively charged colloidal silica attracts Fe³⁺ ions. The resultant reduction in available Fe³⁺ stimulates Fur to unbind from the sip promoter region.