Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Gamma-tsr-1 phage stocks, produced via heat induction, contain a mixture of phages with distinct deoxyribonucleic acid (DNA) content.
• Heterogeneous (HET) fragments in restriction digests of gamma-tsr-1 DNA indicate variations in phage composition, with 20-80% of phages exhibiting these fragments.
• HET fragment appearance correlates with DNA insertions (DI-1) in the gamma phage genome, observed through heteroduplex analysis.

Purpose of the Study:

• To investigate the cause of heterogeneous fragments in gamma-tsr-1 phage DNA.
• To elucidate the mechanism of DNA insertion and the formation of transducing elements in gamma-tsr-1.
• To understand the genetic basis for abnormal phage excision and its link to heat inducibility.

Main Methods:

• Restriction enzyme digestion of gamma-tsr-1 phage DNA.
• Heteroduplex analysis of DNA from gamma-tsr-1 and beta corynebacteriophages.
• DNA-DNA hybridization to identify bacterial DNA insertions.
• Analysis of DNA from phages produced by heat induction versus lytic infection.

Main Results:

• DNA-DNA hybridization confirmed bacterial DNA insertions in the vegetative phage attachment site (attP) of a fraction of gamma-tsr-1 phages.
• These insertions resulted in the formation of HET fragments and were termed transducing elements.
• A second DNA insertion (DI-2) was identified within the tox gene of gamma-tsr-1 and gamma-tsr-2, likely inactivating the gene.

Conclusions:

• Heat-induced gamma-tsr-1 prophage undergoes abnormal, high-frequency excision at a specific site, linked to a mutation in the immune repressor.
• This altered excision specificity, occurring simultaneously with mutation to heat inducibility, distinguishes gamma-tsr-1 transducing element formation.
• The DI-2 insertion in the tox gene suggests a transposition event that inactivated the gene in wild-type gamma phage.