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Improved single-copy integration vectors for Staphylococcus aureus.

ABSTRACT: 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.

SUBMITTER: Luong TT

PROVIDER: S-EPMC2001203 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Improved single-copy integration vectors for Staphylococcus aureus.

Luong Thanh T TT Lee Chia Y CY

Journal of microbiological methods 20070424 1

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 protectio ...[more]

PMID: 17512993

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Project description:BackgroundSingle-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration.FindingsWe have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site.ConclusionsIn this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.

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Improved single-copy integration vectors for Staphylococcus aureus.

Publications

Improved single-copy integration vectors for Staphylococcus aureus.

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