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Characterization and genetic mapping of nontoxinogenic (tox) mutants of corynebacteriophage beta
Abstract:
Seven new nontoxinogenic (tox) mutants of corynebacteriophage beta were isolated. Strains of Cornyebacterium diphtheriae C 7 lysogenic for these tox mutants of beta were tested for their ability to produce extracellular diphtherial toxin or proteins (CRMs) that cross-react immunologically with toxin. By using a sensitive reversed passive hemagglutination assay for toxin antigen, three of the tox mutants were phenotypically CRM+ and four were CRM-. The molecular weights of the CRMs produced by mutants beta tox-1, beta tox-2, and beta tox-3 were determined to be approximately 20,000, 26,000, and 34,000, respectively, by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The 26,000 and 34,000-dalton CRMs had nicotinamide adenine dinucleotide: elongation factor 2 adenosine diphosphate ribose transferase activity, but the 20,000-dalton CRM did not. These three CRMs correspond to amino-terminal fragments of diphtherial toxin and appear to be formed by chain termination during protein synthesis directed by phages with nonsense mutations in the structural gene for diphtherial toxin. No complementation was observed between independently isolated tox mutants of phage beta. The positions of four tox markers on the vegetative genetic map of phage beta were determined, and the orientation of transcription of the structural gene for diphtherial toxin with respect to other markers on the genetic map of phage beta was established.
Insights
Seven new mutants of corynebacteriophage beta were identified. These mutants produced proteins that cross-react with diphtheria toxin, revealing insights into toxin gene structure and function.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- Corynebacteriophage beta is a bacteriophage that infects Corynebacterium diphtheriae.
- The phage carries the gene for diphtheria toxin, a potent exotoxin responsible for the pathogenicity of C. diphtheriae.
Purpose of the Study:
- To isolate and characterize nontoxinogenic mutants of corynebacteriophage beta.
- To investigate the structure and function of diphtheria toxin and its related proteins (CRMs) produced by these mutants.
Main Methods:
- Isolation of seven nontoxinogenic (tox) mutants of corynebacteriophage beta.
- Testing lysogenic C. diphtheriae strains for toxin or CRM production using reversed passive hemagglutination assay.
- Determination of CRM molecular weights by SDS-PAGE.
- Assay of CRM enzymatic activity (nicotinamide adenine dinucleotide: elongation factor 2 adenosine diphosphate ribose transferase).
- Genetic mapping of tox markers on the phage beta genome.
Main Results:
- Three of the seven tox mutants produced CRM+ phenotypes, while four were CRM-.
- CRMs with molecular weights of approximately 20,000, 26,000, and 34,000 daltons were identified.
- The 26,000 and 34,000-dalton CRMs exhibited enzymatic activity, while the 20,000-dalton CRM did not.
- These CRMs represent amino-terminal fragments of diphtheria toxin, likely resulting from premature chain termination due to nonsense mutations.
- No complementation was observed between independently isolated tox mutants.
- The positions of four tox markers and the transcriptional orientation of the toxin gene were mapped on the phage beta genetic map.
Conclusions:
- Nontoxinogenic mutants of corynebacteriophage beta can produce truncated, immunologically cross-reactive proteins (CRMs).
- These CRMs provide evidence for chain termination mutations within the diphtheria toxin structural gene.
- Genetic mapping of these mutants has elucidated the organization and transcriptional direction of the toxin gene within the phage genome.