ABSTRACT
Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Viral , Mycobacteriophages/genetics , Mycobacterium/virology , Repressor Proteins/genetics , Transcription, Genetic , Viral Proteins/genetics , Amino Acid Sequence , Bacteriolysis , Base Sequence , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Bacterial , Lysogeny , Molecular Sequence Data , Mutagenesis , Mycobacteriophages/physiology , Mycobacterium/metabolism , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Repressor Proteins/biosynthesis , Viral Proteins/biosynthesisABSTRACT
The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines.
Subject(s)
BCG Vaccine , Bacteriophages/genetics , Genetic Vectors , Vaccines, Synthetic , Genetic Vectors/genetics , Genome, Viral , Lysogeny , Mycobacterium bovis/virology , Superinfection , Vaccines, AttenuatedABSTRACT
Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. We show here that the 183-amino-acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix DNA-binding motif--properties associated with repressors of temperate phages. We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria. Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic-resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines.