Mutants of the obligate methylothroph Methylobacillus flagellatum KT defective in genes of the ribulose monophosphate cycle of formaldehyde fixation

An approach to the isolation of mutants deficient in key enzymes of C1 metabolism was worked out for the obligate methylotroph Methylobacillus flagellatum KT. The isolation of mutants in genes of the ribulose monophosphate (RMP) cycle of formaldehyde assimilation and oxidation is based on selection of temperature sensitive (ts) mutants followed by enzymatic assays. Standard methods of determination of activities of hexulose phosphate synthase/hexulose phosphate isomerase (HPS/HPI), phosphoglucoisomerase (PGI), glucose 6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (GND) were modified for screening the ts-mutants for the presence of RMP keyenzyme activities. Among the 500 ts-mutants investigated, nine mutants were defective in PGI activity and two in GDP activity. The defective enzymes were characterized by a high rate of inactivation at increased temperatures. At 50° C the proteins studied were completely inactivated during 2 h, whereas the native enzymes maintained more than 80% activity.