Abstract
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.
Highlights
Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis
In 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
For the two phage that were the basis of vectors that we previously developed, the L54a attB site is within the geh gene, whereas the Ø11 attB site is within a hypothetical gene that was previously misannotated to be in an intergenic region [2,18]
Summary
Single-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, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The ability to genetically manipulate bacterial pathogens is essential for the advancement of research on the mechanism of pathogenesis. In order to circumvent these and other problems, we previously developed S. aureus vectors that allow cloning of genes in single copy [2,3] These plasmid vectors are based on the site-specific recombination systems of the lysogenic bacteriophage L54a and Ø11. The vectors can not replicate in S. aureus, but because they each carry a bacteriophage attP site, they can integrate into the bacterial chromosome at the attB site for L54a or Ø11, respectively, in the presence of the bacteriophage integrase (Int) [2,3]
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