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DNA packaging and developmental intermediates of a broad host range Vibrio vulnificus bacteriophage 71A-6
S A Khan1, A A Khan, M S Nawaz
1Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
Abstract:
The structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6, a rod-shaped double-stranded DNA podovirus, were isolated by ultracentrifugation and studied by electron microscopy, SDS-PAGE and pulsed-field gel electrophoretic analysis. Bacteriophage 71A-6 synthesized rod-shaped capsids (mean length=200+/-8 nm; mean width=47+/-3 nm n=50) during its development. Several host proteins that probably help in the assembly and maturation of the capsids were attached to these capsids as spherical structures. A capsid-DNA or DNA packaging complex that consisted of the mature capsids, DNA and a 42.5-kDa protein was also isolated. The size of the capsids increased in length and decreased in width (mean length=220+/-8 nm; mean width=45+/-3 nm n=50) either during or after the DNA packaging. The capsid fractions contained about 12 phage structural proteins and eight host proteins. At least three proteins were tentatively identified: a 38.5-kDa major capsid protein, a 35.2-kDa tail protein and 42.5-kDa packaging initiator or terminator protein. The size of the bacteriophage 71A-6 genome was determined to be 143.0-kb by pulsed-field gel electrophoresis. The total mass of all the mature phage proteins corresponded to only 14.0% of the coding capacity of phage genome.
Insights
Structural intermediates of Vibrio vulnificus bacteriophage 71A-6 were studied. Key proteins involved in capsid assembly and DNA packaging were identified, revealing insights into podovirus replication.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Bacteriophages are viruses that infect bacteria and are crucial in microbial ecosystems.
- Understanding bacteriophage assembly and DNA packaging is vital for phage therapy and genetic engineering.
- Vibrio vulnificus bacteriophage 71A-6 is a double-stranded DNA podovirus with a rod-shaped morphology.
Purpose of the Study:
- To investigate the structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6.
- To identify key phage and host proteins involved in these processes.
- To characterize the morphological changes during DNA packaging.
Main Methods:
- Isolation of structural intermediates via ultracentrifugation.
- Analysis using electron microscopy, SDS-PAGE, and pulsed-field gel electrophoresis.
- Determination of genome size and protein composition.
Main Results:
- Rod-shaped capsids (200 nm length) were synthesized during phage development.
- Capsid dimensions changed (220 nm length) during or after DNA packaging.
- A capsid-DNA complex including a 42.5-kDa protein was isolated; 12 phage and 8 host proteins were identified.
- A 143.0-kb genome was determined, with mature phage proteins comprising only 14.0% of coding capacity.
Conclusions:
- The study elucidated key structural intermediates and protein players in Vibrio vulnificus bacteriophage 71A-6 assembly and DNA packaging.
- Morphological changes in capsids correlate with DNA packaging.
- Identified proteins, including a major capsid protein and a packaging initiator/terminator, are critical for the phage life cycle.