Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

Lloyd M. Kozloff, M. Lute
1981 Journal of Virology  
We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifol4te drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28' or T4D am28). In addition, we found that
more » ... tion, we found that another folate pnalog, pyrimeth4aine, also inhibited T4D 28> production and T4D 28am production, but this na.log did not inhibit wildtype T4D production. A temperatur,-.resistant revertant of T4D 28' was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecularweight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liborated glutamate residues from this substrate with a pH optimum of 8,4 to 8A Eixt-racts of bacteriophage T4D-infected E. coli B cells exibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6,4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfeeted host cells. This new activity was induced in E. coli B cell by infection with wild-type T4D and T4D amber mutants 29-, 26-, 27-, 51-, and 10=, but it was not induced under nonuprmissive conditions by T4D am28 or by T4D 28'. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamnine, whereas the T4D 28' activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wildptype T4D or by T4D gene 28 mutants was alo found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be Inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the fornation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28, Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal suface of the phage baseplate central tail plug. In the accompanying paper (17) Kozloff and plug of the tail baseplate of the phage particle. Zorzopulos present evidence that the product of Previous work (12, 13, 20) indicated that the T4D bacteriophage gene 28 is a protein which gene 28 product plays a catalytic role in cleaving plays a structural role in forming the central very large folyl polyglutamates. When this gene t Pesnt addres: Department of Microbiology, University product was defective, these compounds accuof California, San Francisco, CA 94143. mulated in infected cells. However, a direct dem-645 on May 5, 2020 by guest
doi:10.1128/jvi.40.3.645-656.1981 fatcat:qlxyp5hwcbbjznxryyq2wk2jy4