A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with 3H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.
Spontaneous mutants of Salmonella typhimurium isolated in our laboratory from thiolutin-containing tryptone agar plates are partially resistant to thiolutin in enriched media. In minimal media, they are not resistant. The mutants are not temperature sensitive but fail to support the development of phage P22 at higher temperatures (40 degrees C). Thiolutin did not interfere with RNA polymerase or nucleotide kinase in in vitro experiments. However, thiolutin did inhibit the rate of incorporation of exogenous uridine into the cellular pool and consequently the acid-precipitable material. It appears that one site of action of thiolutin is at the membrane level.
As with other inducible enzymes, the induced synthesis of L-arabinose isomerase (L-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-D-glucose and D-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10-1 M cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3', 5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by D-fucose cannot be reversed by cyclic 3', 5'-AMP. D-Fucose competes with the inducer L-arabinose in some step(s) involved in the process of induction. D-fucose acts as a competitive inhibitor of L-arabinose during the process of induction. MATERIALS AND METHODS Bacterial strain. S. typhimurium LT2 was obtained from Myron Levine of the University of Michigan, Ann Arbor, Mich.
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