A temperature-sensitive expression system based on the Geobacillus stearothermophilus NRS 2004/3a sgsE surface-layer gene promoter

Abstract

The sgsE gene coding for the S-layer (surface layer) protein in the thermophilic Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a is strongly induced when the culture is shifted from optimal (55 degrees C) to maximally tolerable growth temperature (67 degrees C). Here, we investigated the regulation of the sgsE promoter in G. stearothermophilus and tested the function of this promoter in Bacillus subtilis. We used EGFP (enhanced green fluorescent protein) reporter constructs and found that the sgsE promoter has very low basal activity at 28 degrees C, but is approx. 20-fold induced by elevated growth temperatures (37 and 45 degrees C). The promoter confers high expression levels, as EGFP mRNA levels at 45 degrees C were approx. 120-fold more abundant than mRNA levels of the cat (chloramphenicol resistance) gene, which was transcribed from a constitutive promoter on the same plasmid. In fluorescence-microscopic and Western-blot analysis, the EGFP protein was barely detectable at 28 degrees C, whereas intermediate and high levels were detected at 37 and 45 degrees C respectively. The potential to tune expression levels of genes driven by the sgsE promoter in B. subtilis by simple temperature adjustments presents a considerable potential for its future use as high-yield protein expression system for B. subtilis.

Figure 1. Nucleotide sequence of the sgsE 5′-UTR in G. stearothermophilus NRS 2004/3a

Promoters P1/P2 activated in cells grown at 67 °C and promoters P2/P3 activated in cells grown at 55 °C with their −10 and −35 recognition sequences (boldface) are indicated. Transcriptional start points (TSP) are indicted by arrows. RBS, ribosomal binding site. Light grey, IRs (inverted repeats); mid grey, −10 and −35 boxes of promoter P1; dark grey, −10 and −35 boxes of promoters P2 and P3.

Figure 2. qRT–PCR analysis of eGFP gene expression in B. subtilis cells containing a control plasmid lacking the sgsE promoter (negative control, neg.) or containing the sgsE promoter in front of the eGFP coding sequence (P-eGFP)

Cells were pregrown at 37 °C to D₆₀₀ 0.6. Aliquots of the cultures were diluted to D₆₀₀ 0.1 in growth medium and further incubated at 28, 37 or 45 °C until they reached D₆₀₀ 0.8. Equal amounts of RNA were used for transcriptional analysis (for experimental details and normalization of expression levels to cell density and plasmid copy numbers, see the Materials and methods section). eGFP expression levels (expressed as arbitrary numbers on the ordinate axis) were obtained from the normalization procedure.

Figure 3. CLSM images of B. subtilis 168 cells grown at different temperatures

Cells either contained a control plasmid lacking the sgsE promoter in front of eGFP (A) or contained a plasmid expressing eGFP under the control of the sgsE promoter (B–D). (A, D) Cells grown at 45 °C; (B, C) cells grown at 28 and 37 °C respectively. For each panel, a fluorescent picture (left part) and the corresponding non-fluorescent image (right part) are shown. Scale bar, 5 μm.

Figure 4. Western-blot analysis of EGFP expression

B. subtilis cells either containing control constructs (lanes 1–3) or sgsE_p-eGFP constructs (lanes 4–6) were pregrown at 37 °C to D₆₀₀ 0.6. Aliquots of the cultures were diluted to D₆₀₀ 0.1 in growth medium and further incubated at 28 °C (lanes 1 and 4), 37 °C (lanes 2 and 5) or 45 °C (lanes 3 and 6) until they reached D₆₀₀ 0.8. Equal amounts of protein extracts (20 μl of extract; 0.35 mg/ml) were loaded on to the gels. One gel was subjected to Western-blot analysis using anti-GFP antibodies (upper panel) and the second gel was Coomassie Brilliant Blue-stained (lower panel). Strong signals in upper panel (lanes 4 and 5) at ~27 kDa represent EGFP. The values on the right are molecular masses in kDa