Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly.
A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.
Duda
R L
RL
Department of Microbiology, University of California, Los Angeles 90024.
Gingery
M
M
Ishimoto
L K
LK
Eiserling
F A
FA
D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D008854:Microscopy, Electron; D009024:Morphogenesis; D011485:Protein Binding; D013604:T-Phages / Q000235:genetics* / Q000648:ultrastructure; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D014764:Viral Proteins; D015678:Viral Structural Proteins