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Characteristics of phi T, the temperate bacteriophage carried by Bacillus megaterium 899a
Abstract:
Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.
Insights
This study characterizes bacteriophage T, a virus infecting Bacillus megaterium. Researchers detail its plaque formation, DNA density, stability, and burst size, providing insights into phage biology.
Area of Science:
- Microbiology
- Virology
- Molecular Biology
Background:
- Bacteriophages are viruses that infect bacteria and play crucial roles in microbial ecosystems.
- Understanding phage-host interactions is vital for applications in biotechnology and medicine.
- Bacillus megaterium is a model organism for studying bacterial physiology and phage biology.
Purpose of the Study:
- To comprehensively characterize bacteriophage T, a novel phage isolated from Bacillus megaterium.
- To investigate the physical and biological properties of phage T, including its morphology, genetics, and replication cycle.
- To determine the optimal conditions for phage T plaque formation and propagation.
Main Methods:
- Isolation and purification of phage T from Bacillus megaterium lysates.
- Plaque assays under various conditions (nutrient agar, adsorption buffer, temperature, CaCl2).
- Phage DNA extraction and buoyant density centrifugation in CsCl gradients.
- Electron microscopy for phage morphology analysis.
- Burst size determination through phage induction experiments.
Main Results:
- Phage T formed variable plaques on Bacillus megaterium 899a, with clear plaques under optimal conditions.
- Plaque turbidity was influenced by CaCl2 concentration and incubation temperature.
- Phage T DNA has a guanine-cytosine content of 38.2%, with a buoyant density of 1.6975 g/ml.
- The phage exhibited thermal inactivation at 75°C and instability in chloroform.
- Burst sizes varied depending on the host strain and induction conditions, ranging from 47 to 140 phages per cell.
- Electron microscopy revealed a long, noncontracting tail (240 nm) and a hexagonal head (68 x 57 nm).
Conclusions:
- Phage T is a distinct bacteriophage with specific characteristics relevant to Bacillus megaterium.
- The study provides a detailed description of phage T’s physical, chemical, and biological properties.
- Findings contribute to the understanding of phage-host interactions and phage diversity.