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.
abstract: BACKGROUND
  Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB 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 M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Unité de Génétique Mycobactérienne, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France. tvultos@pasteur.fr <tvultos@pasteur.fr>
authors.forename: Tiago Dos
authors.initials: TD
authors.lastname: Vultos
authors.forename: Isabelle
authors.initials: I
authors.lastname: Méderlé
authors.forename: Valérie
authors.initials: V
authors.lastname: Abadie
authors.forename: Madalena
authors.initials: M
authors.lastname: Pimentel
authors.forename: José
authors.initials: J
authors.lastname: Moniz-Pereira
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gicquel
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Reyrat
authors.forename: Nathalie
authors.initials: N
authors.lastname: Winter
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009160:Mycobacteriophages / Q000502:physiology*; D009161:Mycobacterium / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics; D030781:Organisms, Genetically Modified; D012344:RNA, Transfer, Ala / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial / Q000502:physiology; D016662:Virus Integration / Q000502:physiology*
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012344:RNA, Transfer, Ala