Temperature-sensitive mutants of Corynebacterium ammoniagenes ATCC 6872 with a defective large subunit of the manganese-containing ribonucleotide reductase

Abstract

Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides. They were designated nrd ^ts (nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd ^ts mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous growth morphology. In contrast to the wild-type, all three nrd ^ts mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit.