TEMPERATURE-SENSITIVE MUTANTS OF CORYNEBACTERIUM AMMONIAGENES ATCC-6872 WITH A DEFECTIVE LARGE SUBUNIT OF THE MANGANESE-CONTAINING RIBONUCLEOTIDE REDUCTASE

Chemical mutagenesis of the nucleotide-producing strain Corynebacteriu m ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine foll owed by an enrichment protocol yielded 46 temperature-sensitive (ts) c lones. A rapid assay for the allosterically regulated Mn-ribonucleotid e reductase (RRase) was developed with nucleotide-permeable cells of C . ammoniagenes in order to screen for possible defects in DNA precurso r biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, a nd CH 33) grew well at 30 degrees C but did not proliferate at 40 degr ees C because they did not reduce ribonucleotides to 2'-deoxyribonucle otides. They were designated nrrl's (nucleotide reduction defective). When the cultures were shifted from 30 to 40 degrees C, the nrd(ts) mu tants immediately ceased to incorporate radiolabeled nucleic acid prec ursors into the DNA fraction, while DNA chain elongation was barely af fected. Thus, exhaustion of the deoxyribonucleotide pool ultimately in hibited cell division, leading to a filamentous growth morphology. In contrast to the wildtype, all three nrd(ts) mutants displayed a distin ctly enhanced sensitivity of ribonucleotide reduction towards hydroxyu rea (in permeabilized cells and in vitro) at 30 degrees C. The results from assays for biochemical complementation of heat-inactivated (2 mi n, 37 degrees C) mutant enzyme with either the small or the large subu nit of wild-type Mn-RRase located the mutational defect on the large s ubunit.