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

2010; American Society for Microbiology; Volume: 76; Issue: 23 Linguagem: Inglês

10.1128/aem.01671-10

1098-5336

Laetitia Fontaine, Damien Dandoy, Céline Boutry, Brigitte Delplace, Marie Henry de Frahan, Christophe Fremaux, Philippe Horvath, Patrick Boyaval, Pascal Hols,

RNA and protein synthesis mechanisms

A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.