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

¹ Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA
² Department of Biology, Montana Tech, University of Montana, Butte, MT 59701, USA

Correspondence
Graham F. Hatfull
gfh{at}pitt.edu

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.

The GenBank/EMBL/DDBJ accession number for the genome sequence of mycobacteriophage Tweety is EF536069.

Two supplementary figures showing the alignment of Tweety genes 20 and 21 with Che8 gene 2 and confirming a nucleotide deletion in the Tweety genome relative to the homologous segment in phage Llij, a larger version of Fig. 3, and two supplementary tables listing nucleotide repeats in Tweety gene 54 and repeated sequences in phages Tweety gp54, Che8 gp57 and PMC gp51 are available with the online version of this paper.

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