Suppressors of mutations in the bacteriophage T4 gene coding for both RNA ligase and tail fiber attachment activities

D H Hall, R G Sargent, K F Trofatter, D L Russell
1980 Journal of Virology  
The protein product of T4 gene 63 catalyzes both the attachment of tail fibers to fiberless phage particles and the ligation of single-stranded RNA (Snopek et al., Proc. Natl. Acad. Sci. U.S.A. 74:3355-3359, 1977). To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of amM69 in gene 63. We screened for revertants that could grow at 300C but not at 430C on Escherichia coli OK305 when nucleotides were limiting. These false revertants
more » ... e revertants contained the original mutation in gene 63 and new suppressor mutations. Some of these suppressor mutations caused temperature sensitivity by themselves, allowing single mutants carrying the suppressor to be recognized and isolated. The results of mapping and complementation studies indicated that most of these ts suppressors were in the t gene (lysis), one was in gene 5 (baseplate), and one was in gene 18 (sheath). The mutation in gene 18, tsDH638, suppressed three different amber mutations in gene 63 but did not suppress amber mutations in several other genes. None of the suppressors that were characterized were in genes with known functions in nucleotide metabolism. However, an intriguing property of these false revertants was that they were very sensitive to hydroxyurea, an inhibitor of nucleotide metabolism. The protein product of T4 gene 63 catalyzes the last step in phage morphogenesis, the attachment of tail fibers to otherwise complete phage particles (18, 19) . As many as six tail fibers are attached to each tail baseplate by a noncovalent association, and the gene 63 protein does not become part of the structure (16, 18) . Phage with as few as three or four fibers are infectious. This attachment of tail fibers occurs spontaneously (i.e., in the absence of the gene 63 protein) in infected cells at a rate sufficient to produce about 10 infectious phage per cell in a normal infection (18, 19). Snopek et al. (12) have reported that the same gene 63 protein is also an RNA ligase and that the two different activities of this protein differ in requirements and in response to some inhibitors. Several observations are consistent with the suggestion that the gene 63 product might have a role in nucleotide metabolism: (i) the gene 63 product begins to be synthesized early after infection of Escherichia coli (19); (ii) on the genetic map of T4, gene 63 is close to several genes which code for enzymes involved in nucleotide metabolism such as ribonucleotide reductase, thymidylate synthetase, and dihydrofolate reductase (8, 20); and (iii) although gene 63 is nonessential for plaque formation on many strains of E. coli, mutants defective in gene 63 fail to make plaques on E. coli OK305, which is defective in nucleotide metabolism. To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of a gene 63 amber mutant under conditions of nucleotide limitation. These false revertants contain the original mutation in gene 63 and new suppressor mutations that allow bypass of gene 63 function. Characterization of these suppressor mutations has revealed which gene products can be altered to make the gene 63 product unnecessary for plaque formation on E. coli OK305. None of these suppressor mutations are in genes with functions that are related to nucleotide metabolism or to RNA ligation. Our results are consistent with the suggestion that the attachment of tail fibers is the major physiological role of the gene 63 product. MATERIALS AND METHODS Bacteriophage strains. T4Do, an osmotic shockresistant derivative of T4D, was the standard bacteriophage type with which all mutants were compared. T4 amber and temperature-sensitive (ts) mutants (5) were isolated at the California Institute of Technology 103 on May 10, 2020 by guest
doi:10.1128/jvi.36.1.103-108.1980 fatcat:3xfj2pu6lnfe3bo2lnwir7e2pi