Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans / 中华口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system.</p><p><b>METHODS</b>The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette (lox71-Km-lox66), yielding pGEM-T-&Delta;htrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-&Delta;clpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-&Delta;htrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MS&Delta;htrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MS&Delta;htrA, yielding markerless mutant strain lacking clpP and htrA.</p><p><b>RESULTS</b>The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing.</p><p><b>CONCLUSIONS</b>A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.</p>