The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAlagene of Mycobacterium spp.

Abstract

Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attPcontaining 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 tRNAAIa gene. An integration-prof icient plasmid vector was constructed and efficiently inserted at the tRNAAIa gene 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-specif ic integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.

Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

“…Considering the conservative nature of tRNAs, one can expect that the target sites for a particular integrative system could occur in several bacterial species at various levels of relatedness. This view was confirmed by well-documented examples: the site-specific recombination systems of Ms6, mv4, VWB, pSAM2, pSE101, and pSLP1 were shown to function in closely related bacteria (3,5,19,39,51,52). Moreover A2 provides an example of integration in both gram-negative and gram-positive species (2).…”

“…Since the presence of the appropriate target sequence (attB) in the recipient chromosome and specific host factors are required for the integrative process, utilization of a particular integrative recombination system is frequently restricted to the host range of a given phage. A number of known eubacterial temperate phages, such as L5 (37), HP1 (24), 16-3 (35), RP3 (21), U (50), P4 (38), Mx8 (28), Sfi21 (7), 10MC (22), T12 (31), A2 (2), mv4 (16), Ms6 (19), and VWB (51), use tRNA genes as target sequences for integration into the host chromosomes. There are some plasmids with integrative functions, such as pSAM2 (30), pSE101 (5), pSE211 (6), and pSLP1 (52), whose target sites are also within tRNA genes.…”

Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

“…The highest ␤-galactosidase activity was achieved with a plasmid construct, designated pMG1, containing a 252-bp Sau3AI insert. This sequence is located 6 kb upstream of the Ms6 integration locus (6) (Fig. 1A).…”

Plasmidic versus Insertional Cloning of Heterologous Genes inMycobacterium bovisBCG: Impact on In Vivo Antigen Persistence and Immune Responses

“…The pBlaf*-nef-gag cassette was then cut from pJLN29 with ScaI and EcoRV and inserted into the unique EcoRI site of pAV6950 (12), which had been blunt ended with T4 DNA polymerase (Roche-Boehringer) and dephosphorylated with Calf Intestinal Alkaline Phosphatase (Roche-Boehringer). The resulting integration-proficient vectors were called pOIP4 and pOIP12 according to the orientation of the pBlaF*-nef-gag expression cassette in comparison with the Ms6 int gene.…”

“…This could be detrimental to the obtainment of long-term immune memory. Integrative vectors, derived from temperate mycobacteriophages, such as L5 (reviewed in reference 15) or Ms6 (12), have also been developed. These integration-proficient vectors encode integrase functions that allow a recombination event between the phage attP and the bacterial homologous attB sites.…”