Dataset Information

Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.

ABSTRACT: Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions.

SUBMITTER: Pham TT

PROVIDER: S-EPMC2884959 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.

Pham Thuy T TT Jacobs-Sera Deborah D Pedulla Marisa L ML Hendrix Roger W RW Hatfull Graham F GF

Microbiology (Reading, England) 20070801 Pt 8

Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, wit ...[more]

PMID: 17660435

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Project description:BackgroundMycobacteriophage 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.ResultsThe 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.ConclusionSmall 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.

Dataset Information

Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.

Publications

Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.

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