Application of the shsp gene, encoding a small heat shock protein, as a food-grade selection marker for lactic acid bacteria.
Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.
El Demerdash
Hassan A M
HA
Institute for Microbiology, Federal Dairy Research Centre, 24103 Kiel, Germany.
Heller
Knut J
KJ
Geis
Arnold
A
D015415:Biomarkers; D005285:Fermentation; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics*; D010957:Plasmids; D013291:Streptococcus / Q000235:genetics*; D014169:Transformation, Bacterial
D015415:Biomarkers; D006360:Heat-Shock Proteins