| Summary: | <p>Abstract</p> <p>Background</p> <p>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, 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.</p> <p>Findings</p> <p>We 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 <it>Staphylococcus aureus</it>. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (<it>attP</it>) and chromosome (<it>attB</it>) 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 <it>attP </it>site (<it>attP</it>2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified <it>attB </it>site (<it>attB</it>2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in <it>S. aureus </it>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 <it>attB </it>site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site.</p> <p>Conclusions</p> <p>In this work, we describe an <it>E. coli</it>-<it>S. aureus </it>shuttle vector that can be used to introduce any cloned gene into the <it>S. aureus </it>chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of <it>S. aureus </it>and for marking strains for in vivo studies.</p>
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