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Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA
Abstract:
The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973). Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B. subtilis. B. subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake. Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake. Phi W-14 DNA did not alter the kinetics of transfection. The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA. If failed to inhibit when added 30 min after SPP1 DNA. It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA. The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA. Free putrescine did not affect transfection by SPP1 DNA. It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.
Insights
Bacteriophage phi W-14 DNA, uniquely modified with putrescine, significantly inhibits bacterial DNA transfection. This interference occurs intracellularly, suggesting a novel mechanism beyond simple DNA uptake competition.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacteriophage phi W-14 DNA contains alpha-putrescinylthymine, a hypermodified pyrimidine replacing half of its thymine residues.
- Bacterial transformation involves the uptake of exogenous DNA by competent cells.
Purpose of the Study:
- To investigate the effect of bacteriophage phi W-14 DNA on the transfection of Bacillus subtilis by bacteriophage SPP1 DNA.
- To elucidate the mechanism by which phi W-14 DNA influences DNA uptake and transfection.
Main Methods:
- Competent Bacillus subtilis cells were used to study the uptake and transfection of bacteriophage SPP1 DNA.
- The inhibitory effects of Bacillus subtilis DNA and bacteriophage phi W-14 DNA on SPP1 DNA transfection and uptake were compared.
- The timing of DNA addition and concentrations were varied to assess the interaction dynamics.
Main Results:
- Bacteriophage phi W-14 DNA inhibited SPP1 DNA transfection up to 30 times more effectively than it inhibited DNA uptake.
- The inhibition of transfection by phi W-14 DNA was dependent on its addition time relative to SPP1 DNA.
- Intracellular interference was indicated, as free putrescine did not affect transfection, and the basic transfection mechanism remained unchanged.
Conclusions:
- The putrescine groups covalently attached to bacteriophage phi W-14 DNA are responsible for its ability to interfere with the intracellular transfection process.
- Bacteriophage phi W-14 DNA exhibits a unique mechanism of inhibiting DNA transfection, distinct from simple competition for uptake.