A food-grade site-directed mutagenesis system forStreptococcus thermophilusLMG 18311

Abstract

Aims: To develop a general method for site‐directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic‐resistance genes or other selection markers for the identification of transformants. Methods and Results: In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site‐directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony‐lift hybridization using a digoxigenin‐labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation. Conclusions: By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA. Significance and Impact of the Study: A food‐grade site‐directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties.

A 100-Year Review: Yogurt and other cultured dairy products

“…Somkuti and Steinberg (1988) genetically 9991 transformed intact cells of S. thermophilus with plasma DNA by electric field pulses. Blomqvist et al (2010) demonstrated site-directed mutagenesis on S. thermophilus LMG 18311 that could allow construction of starter strains that possess novel or improved properties.…”

Development of a Versatile Procedure Based on Natural Transformation for Marker-Free Targeted Genetic Modification in Streptococcus thermophilus

“…We showed that it can be based on either a two-step procedure consisting of the insertion and subsequent deletion of an antibiotic marker, similar to the procedure for S. mutans (1), or the acquisition/loss of metabolic properties. Blomqvist and coworkers recently proposed a markerless strategy based on colony hybridization with digoxigenin-labeled probes to select small genetic changes in S. thermophilus (6). However, it could turn out to be labor-intensive in the case of strains displaying a transformation rate of Յ10 Ϫ4 , which applies to 4 of 18 strains in our sample of competent strains.…”

“…For S. mutans, the lower competence rate (1%) led to the development of a two-step procedure based on the insertion and subsequent excision of a resistance marker using the CreloxP system (1). For S. thermophilus, some transformation and selection procedures have been proposed previously (6,8). However, they were tested on a limited number of strains, i.e., strains LMD-9, LMG18311, and CNRZ1066, making them difficult to be retained as the most efficient and versatile techniques.…”

Expanding natural transformation to improve beneficial lactic acid bacteria

“…In the context of natural DNA transfers without the use of an antibiotic marker, the major challenges are to use highly transformable strains (Blomqvist et al . 2010 ) or to associate a desired trait with a selectable (growth improvement, color- or texture-marked) phenotype, and to dispose of a natural donor DNA that exhibits a sufficient homology to sustain recombination in the recipient cells. This strategy, reminiscent of self-cloning, was applied to two examples in S. thermophilus , the prtS locus, and the histidine prototrophy.…”