Rhizobium japonicum mutants defective in symbiotic nitrogen fixation

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.