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Acquisition of Thymidylate Synthetase Activity by a Thymine-requiring Mutant of Bacillus subtilis following Infection by the Temperate Phage 3

Microbiology Unit, Department of Biochemistry, University of Oxford

Infection of a thymineless strain of Bacillus subtilis with either the temperate phage 3 or its clear plaque mutant 3c enabled DNA to be formed in the absence of added thymine. Growth of phage 3c under these conditions was associated with an enhanced rate of DNA synthesis compared with uninfected cultures growing in the presence of thymine; when phage 3 became a prophage the lysogenized cells synthesized DNA at the same rate as the uninfected bacteria with thymine. The thymine independence of phage-infected bacteria was due to the acquisition of the enzyme thymidylate synthetase absent in the uninfected mutant bacteria. Phage 3c growth caused a rapid rise in the specific activity of the enzyme after a short lag, and by the time the cells lysed there was approximately ten times more activity than in uninfected wild-type B. subtilis. In thymineless B. subtilis lysogenized with phage 3 the amount of thymidylate synthetase was the same as in wild-type B. subtilis. Phage DNA was able to transform thymine-requiring B. subtilis to thymine independence, showing that it contained the gene for thymidylate synthetase. Plaque-forming particles could not be separated by density gradient centrifugation from those possessing the thymidylate synthetase gene. This result, together with the failure to get ‘thymineless’ mutants of the phage to regain their ability to promote thymine synthesis, suggested that phage 3 was a converting phage. However, the results of transformation implied that the phage thymidylate synthetase gene was closely related to that of the recipient cells, and may have originated from the bacterium.

Received 23 September 1968; accepted 29 October 1968.