%0 Journal Article
%T Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria
%A Tiago Dos Vultos
%A Isabelle M¨¦derl¨¦
%A Val¨¦rie Abadie
%A Madalena Pimentel
%A Jos¨¦ Moniz-Pereira
%A Brigitte Gicquel
%A Jean-Marc Reyrat
%A Nathalie Winter
%J BMC Molecular Biology
%D 2006
%I BioMed Central
%R 10.1186/1471-2199-7-47
%X The three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis 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 tRNAalaU and tRNAalaV T-loops of the same BCG chromosome.Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala 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.Temperate phages integrate into the bacterial chromosome through a site-specific recombination event catalyzed by a phage-encoded recombinase. This process involves a common core present in the phage attP and the bacterial attB genomic DNA sequences, which are identical [1]. Genetic tools based on phage systems have furthered research into the biology of Mycobacterium tuberculosis, a pathogen responsible for about two million deaths each year [2]. L5 [3] and Ms6 [4], both temperate mycobacteriophages, integrate into genes encoding tRNAs. L5 integrates into a tRNAgly gene in the genome of the fast-growing species M. smegmatis or the slow-growing species M. bovis Bacillus Calmette Gu¨¦rin (BCG), wh
%U http://www.biomedcentral.com/1471-2199/7/47