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Escherichia coli capsule bacteriophages. IV. Free capsule depolymerase 29
Abstract:
The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.
Insights
The purified depolymerase from Escherichia coli bacteriophage 29 is identical to free phage 29 spikes. This enzyme degrades host capsule polysaccharides, aiding in phage infection and plaque formation.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacteriophages are viruses that infect bacteria.
- Escherichia coli capsule bacteriophage no. 29 produces a depolymerase enzyme.
- This enzyme causes haloes around phage plaques, indicating host cell lysis.
Purpose of the Study:
- To purify and characterize the depolymerase enzyme.
- To determine if the depolymerase is identical to phage 29 spikes.
- To understand the role of depolymerase in bacteriophage infection.
Main Methods:
- Purification of depolymerase to homogeneity.
- Analysis of molecular weight by sedimentation equilibrium.
- Determination of polypeptide chain sizes using SDS-PAA gel electrophoresis.
- Enzymatic activity assays on host capsular polysaccharide.
- Electron optical imaging for structural comparison.
Main Results:
- Purified depolymerase and phage 29 spikes share identical molecular weight, polypeptide composition, sedimentation coefficient, isoelectric point, and amino acid composition.
- Both depolymerase and phage spikes hydrolyze host capsular polysaccharide into oligosaccharide fragments.
- Phage 29 infection produces significantly more free depolymerase than incorporated spikes.
Conclusions:
- The free depolymerase is composed of phage 29 spikes.
- Phage spikes function as depolymerases, essential for degrading the bacterial capsule.
- This mechanism facilitates phage entry and replication within the host bacterium.