Construction Of The Markless HtrA-Deficient Mutant, Clp-P-Deficient Mutant And HtuA-clpP-Deficient Mutant Of Streptococcus Mutans With Cre-loxP System

Objective Constructing the Streptococcus mutans(S. mutans) htrA-deficient mutant and clpP- deficient mutant with the homologous recombination technique and Cre-loxP site-specific recombination system, then removing the antibiotic resistance marker respectively, to obtain the markerless single gene deficient mutant. These will lay a foundation for the construction of markerless gene deletion mutant strain. To construct markerless double gene deletions at the htrA and clpP loci with the Cre-loxP* system in the basis of Cre-loxP system, which will provide a new method for the construction of markerless multiple gene deletions mutant strain. And the three mutant strains will provide experimental strains for further study of their influences on the cariogenicity of S. mutans.their biological functions in the cariogenic process of S. mutans.Methods 1.A Km resistance gene was amplified by PCR to construct the Km resistance cassette(loxP-Km-lox) where the Km resistance gene was flanked with two loxP site. The DNA fragment containing htrA was amplified by PCR and cloned into the pGEM-T-Easy TA cloning vector.Then the recombinant plasmid was digested and linked with the Km resistance cassette, yielding homologous recombination vector pIBâ–³htrA-Km. Electrotransformation of S. mutans cells with pIBâ–³htrA-Km resulted in isolation of Km resistant S. mutans transformants.2.A Sp resistance gene was amplified by PCR to construct the Sp resistance cassette(loxP-Sp-loxP) where the Sp resistance gene was flanked with two loxP site. After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector, it was digested and linked with the Sp resistance cassette, yielding homologous recombination vector pIBâ–³clpP-Sp. The vector was used for the transformation of S. mutans, with transformants selected on TPY plates containing Sp.3.The Km resistant S. mutans transformants and the Sp resistant S. mutans transformants were transformed with the thermosensitive plasmid pCrePA respectively to excise the Km and the Sp resistance gene. The pCrePA was then easily eliminated at nonpermissive temperature, resulting in a markerless mutant strain carrying a deletion at the htrA loci and a markerless mutant strain carrying a deletion at the clpP loci, which were verified by PCR and DNA sequencing. 4.Constructing lox71-Km-lox66 and lox71-Sp-lox66, to replace loxP-Km-loxP and loxP-Sp-loxP separately. Using the above method, to constructe the markerless htrA-deficient mutant.Then, the clpP and Sp resistance gene were deleted from such a mutant, yielding markerless mutant strain lacking clpP and htrA, which was verified by PCR and DNA sequencing.Result 1.The DNA fragments including htrA and clpP, the Km resistance gene and the Sp resistance gene were amplified by PCR. The PCR products show a single Bright band analyzing through SDS-PAGE, non-specific amplification, molecular weight are the same as expected.2.The constructions of all the recombinant plasmids with expected open frame were confirmed though restriction enzyme mapping analysis. The E.coli containing Km-resistant plasmid and Sp-resistant plasmid could grow well in LB medium with Km resistance and Sp resistance respectively. These antibiotic resistance genes could be expressed in vitro.3.The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing. There was a loxP at this loci in single gene deficient mutant, and a Iox72 in the double gene deficient mutant.Conclusion The markless htrA-deficient mutant, the markerless clpP-deficient mutant, the markerless double gene deletions at the htrA and clpP loci were constructed successfully utilizing the homologous recombination technique and Cre-loxP system, which provide a new and effective method for generating markerless multiple gene deletion mutants. In addition, these provide experimental strains for further study of the biological functions of the htrA and clpP gene.