Screening for mutants of Rhizobium japonicum with defects in nitrogen fixing ability

Mutants of Rhizobium japonicum with reduced ex planta nitrogenase activity could be isolated with high frequency by direct screening of ultra-violet mutagenized bacteria growing as spots on the surface of an appropriate agar medium permitting derepression of nitrogenase synthesis. Small glass chambers fitted with a serum cap were pushed into the agar around each spot of growth, forming a small enclosed gas space which was made to 10% acetylene, permitting assessment of nitrogenase activity by the acetylene reduction test. Four mutants were isolated from a total of 305 screened spots. Two mutants had almost no ex planta activity, one of which had no symbiotic activity despite normal nodulation (ineffective), the other had only somewhat reduced activity symbiotically. Two other mutants with less than half wild-type activity ex planta were normal in symbiosis.