Cloning and heterologous expression of the plasmidencoded shsp gene of Streptococcus thermophilus isolated from Chinese dairy
We first tested 12 strains of Streptococcus thermophilus isolated from China yogurt or its starter culture for their plasmid content. Two strains were found to harbor two plasmids, identified as pQC1 and pQC2, from the S. thermophilus ST-QC, as well as pHS1 and pHS2 from the S. thermophilus ST-HS. Agarose gel electrophoresis profiles indicated that the molecular size of about 4.5 kb for the two bigger plasmids (pQC1 and pHS1) was nearly identical, same with the molecular size of approximately 3.5 kb for the two smaller plasmids (pQC2 and pHS2). A 765 bp DNA fragment, including a 429 bp open reading frame of the shsp gene of the smaller plasmid pQC2 from S. thermophilus St-QC, was successfully cloned and sequenced. Multiple sequence alignment revealed that the nucleotide sequence of the coding region of the shsp gene or the deduced amino acid sequence of the sHSP protein shared a high degree of identity (> 86.67 or 81.33% identity) with the shsp genes or the sHSP proteins described from other S. thermophilus plasmids. In addition, 222 bp nucleotide sequences upstream and 114 bp nucleotide sequences downstream belonging to the shsp gene coding region were also analyzed. The separation of SDS-PAGE and the analysis of Western blotting for the soluble cell proteins showed that the shsp gene of plasmid pQC2 of S. thermophilus St-QC was successfully expressed in mesophilic Escherichia coli. In addition to strong heat and acid tolerance, recombinant E. coli cells overexpressing the S. thermophilus St-QC shsp gene had significantly higher resistance to ethanol stress, which is the first physiological function found to be linked to the S. thermophilus plasmid-borne shsp gene. This study will provide a basis for the cloning and expression of the shsp genes from a thermophilic microorganism in the mesophilic LAB or yeast and for further development of stress-resistant microorganism strains used in dairy fermentation and brewing wine.
Key words: Streptococcus thermophilus, plasmid, small heat shock protein gene (shsp gene), cloning, expression, abiotic stresses, Escherichia coli.