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Microbiology 144 (1998), 3397-3406; DOI  10.1099/00221287-144-12-3397
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The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAlagene of Mycobacterium spp.

Acilino Freitas-Vieira, Elsa Anes and J. Moniz-Pereira

Laboratório de Microbiologia Molecular, Faculdade de Farmácia, Universidade de Lisboa, Av. das Forças Armadas, 1600 Lisboa, Portugal

ABSTRACT

Summary: Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5' end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3' end of the tRNAAlagene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNAAlagene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.

Author for correspondence: J. Moniz-Pereira. Tel: +351 1 7946430. Fax: +351 1 7934212. e-mail: jpereira@ff.ul.pt

Keywords: Mycobacterium phage, site-specific recombination, tRNAAla, integrative vectors




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