Cloning of the nrd Gene Encoding Nucleotide Reductase in Salmonella typhimurium

다국어 초록 (Multilingual Abstract)

The Salmonella typhimurium nrd gene encoding ribonucleotide reductase (EC 1.17.4.1) was cloned by shotgun complementation of the nrdAB mutations in E. coli KK535. The derived pSK1 containing the 3.8 kb PstI fiagment of S. typhimurium chromosome was selected. For complementation of the temperature sensitive nrdA mutation, a whole stretch of the insert was required. When the E. coli KK535/pSKI was cultured at a non-permissive temperature, the nucleotide reductase activity was doubled in the late-logarithmic phase compared to the cells cultured at the permissive temperature, however, the activity of the cells cultured at both temperatures paralleled each other in the stationary phase. The enzyme consisted of a 58 kDa monomer in the minicell experiment. By chelating it with EDTA, the activity decreased significantly, but other agents like Na-azide, para-chloromercucibenzoate, mercaptoethanol, and urea, were not as effective. Nucleoside diphosphate substrates were reduced more efficiently than triphosphates and the coenzyme B_(12) acted as a cofactor. Considering these results, the cloned Salmonella nrd gene could not be categorized with the three known types of nucleotide reductases.