Under iron-limiting conditions, many bacteria secrete ferric iron-specific ligands, generically termed siderophores, to aid in the sequestering and transport of iron. One strain of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, 61A152, was shown to produce a siderophore when 20 B. japonicum strains were screened with all six chemical assays commonly used to detect such production. Production by strain 61A152 was detected via the chrome azurol S assay, a general test for siderophores which is independent of siderophore structure. The iron-chelating compound was neither a catechol nor a hydroxamate and was ninhydrin negative. It was determined to be citric acid via a combination of thin-layer chromatography and high-voltage paper electrophoresis; this identification was verified by a specific enzymatic assay for citric acid. The inverse correlation which was observed between citric acid release and the iron content of the medium suggested that ferric citrate could serve as an iron source. This was confirmed via growth and transport assays. Exogenously added ferric citrate could be used to overcome iron starvation, and iron-deficient cells actively transported radiolabeled ferric citrate. These results, taken together, indicate a role for ferric citrate in the iron nutrition of this strain, which has been shown to be an efficient nitrogen-fixing strain on a variety of soybean cultivars.Iron-containing proteins figure prominently in the nitrogen-fixing symbioses between bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium and their respective plant hosts. Nitrogenase can constitute 10 to 12% of the total protein in a bacterial cell and leghemoglobin may represent as much as 25 to 30% of the total soluble protein in infected plant cells (47). For the synthesis of these and other iron-containing compounds, such as ferredoxin, hydrogenase, and cytochromes, plants and bacteria must have an adequate supply of iron. This is well illustrated by the report that iron deficiency specifically limits nodule development in peanut plants inoculated with Bradyrhizobium sp. (29). Iron-stressed plants had fewer bacteroids present in the nodules, showed decreased amounts of leghemoglobin, and had lower specific nitrogenase activity on a fresh-weight basis (29). Additional evidence for the importance of iron in nitrogen-fixing symbioses is provided by a mutant of Rhizobium leguminosarum which forms white, ineffective nodules on peas and has an apparent defect in iron acquisition (24). These in planta results complement previous work on the effects of iron deficiency on free-living rhizobia (36). Cultures of irondeficient cells had decreased cell yield, decreased cytochrome content, and diminished activity for the first two enzymes in the heme biosynthetic pathway (36). In addition to these reports, there is new evidence that iron plays a regulatory role in the nitrogen fixation process (11,14)

Bradyrhizobium japonicum USDA 110 and 61A152 can utilize the hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type siderophore pseudobactin St3. The ability to utilize another organism's siderophores may confer a selective advantage in the rhizosphere.