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Penetration of a bacteriophage into Bacillus subtilis: blockage of infection by deoxyribonuclease
Abstract:
Plaquing of a newly isolated phage of Bacillus subtilis, phage 41c, is only 2% efficient in agar containing 200 mug of deoxyribonuclease per ml. Timed deoxyribonuclease addition experiments showed that phage development is blocked in 90% of the cells if deoxyribonuclease is present during adsorption (zero-time samples), whereas 10 min after adsorption the enzyme has little effect (10-min samples). The fate of (32)P-deoxyribonucleic acid label of phage 41c in zero-time samples was compared to that in 10-min samples. In both, about 80% of the label remained with the phage-bacterium complex on initial centrifugation. However, four successive washings removed 90% of the (32)P from the zero-time samples but only 25% from the 10-min samples. In both samples, most of the washed-out label was of low molecular weight. When the time course of interruption of infection by blending was compared with interruption by deoxyribonuclease treatment, the two processes exhibited similar kinetics. It is postulated that both processes block injection at the same site, namely, the point of contact between phage tail and cell wall surface. Partitioning of (32)P label during protoplasting of zero-time and 10-min samples was similar to that observed during washing. For the protoplasting experiments, a quantitative method for plaquing protoplasts was developed. A single bacillus made of several cells can give rise to several protoplast plaque-forming units. Strain 41c was the only phage of seven tested to be inhibited by deoxyribonuclease. No other deoxyribonuclease-sensitive phages have been described.
Insights
Deoxyribonuclease inhibits Bacillus subtilis phage 41c development by blocking injection, particularly when added during adsorption. This phage-bacterium interaction is sensitive to deoxyribonuclease, unlike other tested phages.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophages are viruses that infect bacteria.
- Bacillus subtilis is a common soil bacterium.
- Phage-bacterium interactions are crucial in microbial ecology and biotechnology.
Purpose of the Study:
- To investigate the effect of deoxyribonuclease on the plaquing efficiency of a newly isolated Bacillus subtilis phage, designated 41c.
- To determine the stage of phage development sensitive to deoxyribonuclease.
- To elucidate the mechanism by which deoxyribonuclease inhibits phage infection.
Main Methods:
- Plaquing assays with varying deoxyribonuclease concentrations.
- Timed deoxyribonuclease addition experiments.
- Radioactive labeling of phage deoxyribonucleic acid with 32P.
- Centrifugation, washing, and protoplasting techniques.
- Comparison with blending as an interruption method.
Main Results:
- Phage 41c plaquing efficiency was significantly reduced (98%) in the presence of deoxyribonuclease.
- Deoxyribonuclease inhibited phage development when present during adsorption but had minimal effect when added 10 minutes post-adsorption.
- 32P-labeled phage deoxyribonucleic acid was rapidly released from the phage-bacterium complex in the presence of deoxyribonuclease during adsorption, indicating blocked injection.
- Blending and deoxyribonuclease treatment showed similar kinetics for interrupting infection, suggesting a common inhibition site.
Conclusions:
- Deoxyribonuclease specifically inhibits Bacillus subtilis phage 41c infection by blocking the injection of its genetic material.
- The point of phage tail-cell wall contact is postulated as the common site of inhibition for both deoxyribonuclease and blending.
- Phage 41c is uniquely sensitive to deoxyribonuclease among the seven phages tested, suggesting novel mechanisms of interaction or entry.