A Rhizobium leguminosarum mutant defective in symbiotic iron acquisition.

Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically. These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R. leguminosarum that is essential for symbiotic nitrogen fixation.

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.

Stimulation of tetrapyrrole formation in Rhizobium japonicum by restricted aeration.

Cultures of Rhizobium japonicum were grown with vigorous aeration to stationary phase and were then incubated under restricted aeration for several days. Under these "microaerobic" conditions, cellular heme content increased 10-fold, and visible amounts of porphyrins were released into the culture medium. The two predominant porphyrins produced were identified, on the basis of their spectrophotometric and chromatographic properties, as protoporphyrin and coproporphyrin. The cytochrome complement of microaerobic cells partially resembled that of the symbiotic bacteria in that cytochromes alpha-alpha3 were absent and a CO-binding cytochrome 552 was present. During the period of restricted aeration, at the time that the heme content was increasing, there was a similar 10-fold increase in the activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase. However, during the same period, the activity of succinyl thiokinase (an enzyme that is required in large amounts whether or not heme is being produced) increased only twofold. These results suggest that reduced oxygen tension may play a role in inducing heme synthesis necessary for leghemoglobin formation and bacterial differentiation in soybean root nodules.

Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta-Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin.

During nodulation of soybean (Glycine max) by Rhizobium japonicum, variations in the activities of two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) and delta-aminolevulinic acid dehydrase (ALAD) are described. delta-Aminolevulinic acid synthase activity is found in the bacteroid fraction of nodules, but is not detected in the plant fraction. Bacteroid ALAS activity parallels heme accumulation during nodule development. delta-Aminolevulinic acid dehydrase activity is found in both bacteroid and plant cytosol fractions. Bacteroid ALAD activity is constant or increases during nodulation while plant ALAD activity falls.Bacteroid ALAD activity is found in effective, not in inefficient nodules. Plant ALAD activity is found in both effective and inefficient nodules. Plant ALAD activity falls during development of both types of root nodules.These results support the contention that it is the bacteroid ALAS and ALAD activities, not those of the plant, that are directly involved in formation of leghemoglobin heme, suggesting that the bacteroid may be solely responsible for formation of leghemoglobin heme in the nodule symbiosis.

Calcium Requirement for Indoleacetic Acid-induced Acidification by Avena Coleoptiles.

The ionic specificity of IAA-induced acidification by Avena coleoptiles was studied, using zwitterionic, presumably impermeant buffers. The acidification was almost totally dependent on divalent cations with an order of effectiveness of Ca(2+) >/= Sr(2+) > Mn(2+), Mg(2+); whereas other polyvalent cations tested were ineffective. The Ca(2+) response was IAA-dependent. The CaCl(2) concentration was optimal at 0.3 to 1 mm and inhibitory at higher concentrations. Sr(2+) inhibited Ca(2+)-dependent acidification and monovalent cations such as K(+) did not induce additional acidification in the presence of optimal CaCl(2). These data are consistent with a mechanism for IAA-induced acidification involving a Ca(2+) -H(+) exchange.

Development of chlorophyll and hill activity.

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding delta-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with delta-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.We conclude that the development of photosystem II is limited by the synthesis of proteins in both the cytoplasm and the plastid, not by chlorophyll synthesis. Prolonged illumination is necessary for the development of oxygen evolution.