{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T22:59:08Z","timestamp":1762297148897},"reference-count":21,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Molecular Biol"],"published-print":{"date-parts":[[2006,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>Mycobacteriophage Ms6 integrates into <jats:italic>Mycobacterium smegmatis<\/jats:italic> and <jats:italic>M. bovis<\/jats:italic> BCG chromosome at the 3' end of tRNA<jats:sup>ala<\/jats:sup> genes. Homologous recombination occurs between the phage <jats:italic>attP<\/jats:italic> core and the <jats:italic>attB<\/jats:italic> site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in <jats:italic>M. smegmatis<\/jats:italic> and BCG. We then aimed to modify the <jats:italic>attP<\/jats:italic> site in Ms6-derived vectors, to switch integration to other tRNA<jats:sup>ala<\/jats:sup> loci. This provided the basis for the development of recombinant <jats:italic>M. bovis<\/jats:italic> BCG strains expressing several reporter genes inserted into different tRNA<jats:sup>ala<\/jats:sup> genes.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The three tRNA<jats:sup>ala<\/jats:sup> genes are highly conserved in <jats:italic>M. smegmatis<\/jats:italic> and BCG. However, in the T-loop of tRNA<jats:sup>alaU<\/jats:sup> and tRNA<jats:sup>alaV<\/jats:sup> containing the <jats:italic>attB<\/jats:italic> site, a single base difference was observed between the two species. We observed that the tRNA<jats:sup>alaU<\/jats:sup> gene was the only site into which Ms6-derived integration-proficient vectors integrated in <jats:italic>M. smegmatis<\/jats:italic>, whereas in BCG, the tRNA<jats:sup>alaV<\/jats:sup> gene was used as the target. No integration occurred in the BCG tRNA<jats:sup>alaU<\/jats:sup> T-loop, despite a difference of only one base from the 26-base Ms6 <jats:italic>attP<\/jats:italic> core. We mutated the <jats:italic>attP<\/jats:italic> core to give a perfect match with the other tRNA<jats:sup>ala<\/jats:sup> T-loops from <jats:italic>M. smegmatis<\/jats:italic> and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNA<jats:sup>alaU<\/jats:sup> and tRNA<jats:sup>alaV<\/jats:sup> T-loops of the same BCG chromosome.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>Small changes in the 7 bp T-loop <jats:italic>attP<\/jats:italic> site of Ms6 made it possible to use another <jats:italic>attB<\/jats:italic> site, albeit with a lower integration efficiency. These molecular studies on BCG tRNA<jats:sup>ala<\/jats:sup> genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2199-7-47","type":"journal-article","created":{"date-parts":[[2006,12,15]],"date-time":"2006-12-15T19:16:53Z","timestamp":1166210213000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria"],"prefix":"10.1186","volume":"7","author":[{"given":"Tiago","family":"Dos Vultos","sequence":"first","affiliation":[]},{"given":"Isabelle","family":"M\u00e9derl\u00e9","sequence":"additional","affiliation":[]},{"given":"Val\u00e9rie","family":"Abadie","sequence":"additional","affiliation":[]},{"given":"Madalena","family":"Pimentel","sequence":"additional","affiliation":[]},{"given":"Jos\u00e9","family":"Moniz-Pereira","sequence":"additional","affiliation":[]},{"given":"Brigitte","family":"Gicquel","sequence":"additional","affiliation":[]},{"given":"Jean-Marc","family":"Reyrat","sequence":"additional","affiliation":[]},{"given":"Nathalie","family":"Winter","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2006,12,15]]},"reference":[{"key":"136_CR1","doi-asserted-by":"crossref","first-page":"7495","DOI":"10.1128\/jb.174.23.7495-7499.1992","volume":"174","author":"AM Campbell","year":"1992","unstructured":"Campbell AM: Chromosomal insertion sites for phages and plasmids. 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