We have previously reported the construction of Staphylococcus aureus single-copy integration vectors based on the lysogenic bacteriophage L54a site-specific recombination system. These vectors can replicate autonomously in Escherichia coli, which allows DNA manipulations. In S. aureus, the vectors, which do not possess staphylococcal replication function, can only be maintained by integrating into the chromosome. However, the original vectors have limited cloning sites and do not have protection from potential transcription of external promoters. Here we report the improved version of these vectors that circumvent these shortcomings. In addition, a second integration site based on the bacteriophage phi11 site-specific recombination system has been added such that the vectors can integrate either at the L54a attachment site or at the phi11 attachment site.
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