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

Abstract

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.

Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

“…Using luciferase reporter strains and transcriptome comparisons, we demonstrate that the inactivation of MecA has no effect on the expression of early com genes such as comX but largely affects the expression of late com genes. However, the inactivation of mecA is insufficient to activate natural transformation under these conditions (data not shown), while the overexpression of comX led to a low but detectable transformation rate (16). Similarly, no improvement in the transformation rate of a ClpC-deficient strain of LMG18311 grown under nonpermissive THBG conditions was reported (1).…”

“…In a second set of experiments, the functional role of MecA from LMD-9 and LMG18311 strains was evaluated by growing cells in CDML medium. These growth conditions were shown previously to be permissive for spontaneous competence development at high and low levels for LMD-9 and LMG18311, respectively (16).…”

“…LMD-9 derivative strains CB0011, CB0071, and CB0051 and LMG18311 derivative strain CB0061 were constructed by replacing the sequence between the start and stop codons of mecA with the chloramphenicol resistance cassette lox66-P32-cat-lox71 (mecA::lox66-P32-cat-lox71) according to procedures described previously by Fontaine et al (16). The primers used to construct these strains are listed in Table S1 in the supplemental material.…”

“…Plasmid pMGXstrep was electroporated into wild-type (WT) strain LMD-9 and its derivative strains deficient in MecA, ClpC, and ClpE (CB0051, AW001, and AW003, respectively). Lactococcus lactis overproducing ComXStrepTagII (A. Wahl, unpublished data) and WT LMD-9 carrying plasmid pMGX (producing ComX without StrepTagII) (16) were used as positive and negative controls, respectively. These strains were grown overnight in THBG containing 5 g ml Ϫ1 erythromycin at 37°C.…”

“…Strains CB0012, CB0072, and CB0052 were constructed by excising the chloramphenicol marker from strains CB0011, CB0071, and CB0051, respectively, using the Cre-loxP system. This procedure was performed by using plasmid pGhostcre as described previously (16).…”