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.

Fig. 1.

Virion morphology of Tweety. Electron micrograph of mycobacteriophage Tweety particles. Scale bar, 100 nm.

Fig. 2.

Nucleotide sequence comparisons of the Tweety, Che8, Che9d, Llij and PMC genomes. The extent of DNA sequence similarity among these mycobacteriophage genomes is illustrated in a Dotter plot using a sliding window of 25 bp (Sonnhammer & Durbin, 1995).

Fig. 3.

Map of the Tweety genome and comparison to maps of Che8, Llij, PMC and Che9d. Genomes are represented by horizontal lines with putative genes shown as boxes above (transcribed rightwards) or below (transcribed leftwards) each genome; the number of each gene is shown within each box. The diagonal arrow indicates a programmed translational frameshift between Tweety genes 12 and 13. All genes have been assorted into phamilies (Phams) of related sequences using the computer program ‘Phamerator’ (S. Cresawn, R. W. Hendrix & G. F. Hatfull, unpublished data); the phamily number is displayed above each gene and the boxes colour-coordinated accordingly. Note that the Pham numbers differ from those described previously (Hatfull et al., 2006). Putative gene functions are noted. (A larger version of this figure is available as supplementary data with the online version of this paper.)

Fig. 4.

An unusual repeated sequence within Tweety gene 54. (a) Plot of mol% G+C across the Tweety genome, revealing a region of very high mol% G+C within gene 54. The approximate positions where changes in mol% G+C occur are indicated. (b) Alignment of Tweety gp54, Che8 gp57 and PMC gp51, with amino acid identities shown by asterisks. Conserved substitutions are indicated by colons, and semi-conserved substitutions by periods. The red box indicates the amino acid sequence of Tweety gp54 that corresponds to the segment of high mol% G+C in panel a. (c) Sequence of Tweety gp54 showing the locations of repeated sequences. The repeats can be organized as octapeptide repeats (shown as alternating green and red boxes), or as tetrapeptide repeats (shown as alternating darker- and lighter-coloured boxes). Alignments of the sequences of the nucleotide and tetrapeptide repeats are shown in Supplementary Tables S1 and S2 respectively.

Fig. 5.

Integration of Tweety into the M. smegmatis genome. (a) Sequence alignment of a segment of the Tweety genome immediately upstream of the int gene (43) (coordinates 32 362–32 290) with part of the M. smegmatis genome (coordinates 4 847 908–4 847 991) reveals a common core sequence (boxed); two non-identical positions are shown in bold type. The position of the M. smegmatis tRNA^Lys gene (Msmeg 4746) is indicated by the arrow. (b) Structure of tRNA^Lys, with an arrow indicating the position corresponding to the 5′ position of the common core. Positions that change following Tweety integration are shown in bold. (c) Organization of integration-proficient plasmid pTT1B and its integration into the M. smegmatis chromosome.