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Applied and Environmental Microbiology, August 2003, p. 4408-4412, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4408-4412.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

Hassan A. M. El Demerdash, Knut J. Heller,^* and Arnold Geis

Institute for Microbiology, Federal Dairy Research Centre, 24103 Kiel, Germany

Received 3 September 2002/ Accepted 17 May 2003

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°C or pH 3.5 and in the ability to grow at 52°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 10⁴ and 1.0 x 10⁴ CFU/0.5 µg of DNA, with standard deviations of 0.54 x 10⁴ and 0.32 x 10⁴, for shsp and Em^r selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10⁴ and 3.8 x 10³ CFU/0.5 µg of DNA, with standard deviations of 0.63 x 10⁴ and 3.48 x 10³, 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.

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* Corresponding author. Mailing address: Institute for Microbiology, Federal Dairy Research Centre, P.O. Box 6069, D-24121 Kiel, Germany. Phone: 49(0)431-6092340. Fax: 49(0)431-6092306. E-mail: heller{at}bafm.de.

Present address: Biotechnology Research Center, Suez Canal University, Ismailia 41522, Egypt.

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Applied and Environmental Microbiology, August 2003, p. 4408-4412, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4408-4412.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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