Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.
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.
Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Thuy T
TT
Pham
Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Deborah
D
Jacobs-Sera
Department of Biology, Montana Tech, University of Montana, Butte, MT 59701, USA.
Marisa L
ML
Pedulla
Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roger W
RW
Hendrix
Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Graham F
GF
Hatfull
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics; D008242:Lysogeny; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D020102:Mycobacterium smegmatis / Q000821:virology*; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial; D014771:Virion / Q000648:ultrastructure
D004279:DNA, Viral; D019426:Integrases