Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus

Abstract

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.

FIG. 1.

Expression of the luc reporter gene (▴) during growth of S. thermophilus LMG 18311 from logarithmic to stationary phase (▪). (A) Expression of luciferase driven by the comX promoter. (B) Expression of luciferase driven by the comEC late gene promoter.

FIG. 2.

Effect of the growth medium on STP-induced overexpression of the luciferase reporter protein. Expression of the luc reporter gene was driven by the promoter of the putative bacteriocin gene, stbD. The S. thermophilus BP strain was grown in THG (blue lines) or HJGL (yellow lines) from logarithmic to stationary phase. The STP peptide pheromone was added at time zero.

FIG. 3.

Transcriptional activation of late competence genes by STP-induced overexpression of ComX in S. thermophilus LMG 18311 carrying the pXL plasmid. Plasmid pXL contains an expression cassette consisting of the STP-responsive stbD promoter transcriptionally fused to the comX gene. In addition, it contains a reporter cassette consisting of the comEC late gene promoter transcriptionally fused to the luc reporter gene. As ComX activates transcription from late gene promoters, this construct makes it possible to monitor expression of late competence genes in STP-induced (▴) and uninduced (▵) S. thermophilus cultures. ▪, growth curve for STP-induced culture; □, growth curve for uninduced culture. The STP peptide pheromone was added at time zero.