A temperature-sensitive expression system based on the Geobacillus stearothermophilus NRS 2004/3a sgsE surface-layer gene promoter.
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.
Novotny
Rene
R
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria.
Berger
Harald
H
Schinko
Thorsten
T
Messner
Paul
P
Schäffer
Christina
C
Strauss
Joseph
J
D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D017930:Genes, Reporter; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013696:Temperature
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins