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.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia.

A 200-megadalton plasmid was mobilized from Rhizobium japonicum USDA 191 to other Rhizobium strains either that cannot nodulate soybeans or that form Fix- nodules on certain cultivars. The symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.     

Transformation in Rhizobium japonicum

Summary The transformation of streptomycin resistance in Rhizobium japonicum was studied. The susceptible strain 211 was selected from sixty strains and one step mutant resistant to streptomycin in concentration 1 mg per 1 ml was used as the donor. The peak of the competence curve appeared at the ninth hour of growth; the frequency, when the homologous strain had been used was 0.01 p.c. The transformed resistance was of the same level as in the donor strain.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes).     

L-Arabinose metabolism in Rhizobium japonicum

l-Arabinose was metabolized through an oxidative pathway by extracts of a strain of Rhizobium japonicum. The findings showed that l-arabinose is converted into 2-keto-3-deoxy-l-arabonate, which is cleaved into glycoaldehyde and pyruvate.     

Cytokinin Production by Rhizobium japonicum

Possible hormonal interactions between soybean roots and the Rhizobium initiating nodule proliferation in this genus were studied. A cytokinin has been isolated by column and paper chromatography from an effective strain of Rhizobium japonicum grown in pure culture. The substance promotes cell proliferation in a cytokinin-requiring soybean callus tissue. The bacteria are capable of conditioning a cylokinin-free soybean culture medium so that it is able to support the cytokinin-requiring tissue. It is concluded that the substance is a product of the bacteria rather than an artifact of purification. This unidentified cytokinin or a substance moving to a similar Rf value on the paper chromalogram produces polyploid divisions when tested on cultured pea root segments. Some of the division figures exhibit the diploclirornosomes typical of the root nodule primordium in pea.     

Gluconate Catabolism in Rhizobium japonicum

Gluconate catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled gluconate gave the following results for growing cells, with values expressed as per cent (14)CO(2) evolution: C-1 = 93%, C-2 = 57%, C-3 = 30%, C-4 = 70%, C-6 = 39%. The preferential release of (14)CO(2) from C-1 and C-4 indicate that gluconate is degraded primarily by the Entner-Doudoroff pathway but the inequalities between C-1 and C-4 and between C-3 and C-6 indicate that another pathway(s) also participates. The presence of gluconokinase and a system for converting 6-phosphogluconate to pyruvate also indicate a role for the Entner-Doudoroff pathway. The extraordinarily high yield of (14)CO(2) from C-1 labeled gluconate suggests that the other participating pathway is a C-1 decarboxylative pathway. The key enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, could not be demonstrated. Specifically labeled 2-ketogluconate and 2,5-diketogluconate were oxidized by gluconate grown cells and gave ratios of C-1 to C-6 of 2.73 and 2.61, respectively. These compare with a ratio of 2.39 obtained with specifically labeled gluconate. Gluconate dehydrogenase, the first enzyme in the ketogluconate pathway found in acetic acid bacteria, was found. Oxidation of specifically labeled pyruvate, acetate, succinate, and glutamate by gluconate-grown cells yielded the preferential rates of (14)CO(2) evolution expected from the operation of the tricarboxylic acid cycle. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the primary pathways of gluconate oxidation in R. japonicum. An ancillary pathway for the initial breakdown of gluconate would appear to be the ketogluconate pathway which enters the tricarboxylic acid cycle at alpha-ketoglutarate.     

Tryptophan auxotrophs of Rhizobium japonicum

Eleven tryptophan-requiring mutants of Rhizobium japonicum I-110 ARS were isolated after nitrous acid mutagenesis and fell into five groups based on characterization by supplementation with intermediates and enzyme assays.     

Streptomycin resistance in Rhizobium japonicum

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.     

Glucose Catabolism in Rhizobium japonicum

Glucose catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled glucose gave the following results for resting cells, with values expressed as per cent (14)CO(2) evolution: C-1=59%, C-2=51%, C-3=45%, C-4=59%, and C-6=43%. These values indicate that glucose was degraded by the Entner-Doudoroff pathway alone. Cells which grew in glucose-yeast extract-salts medium gave essentially the same pattern except for retardation of the C-6 carbon. The rates were: C-1=54%, C-2=42%, C-3=51%, C-4=59%, and C-6=32%. Hexokinase, glucose-6-phosphate dehydrogenase, transketolase, and an enzyme system which produces pyruvate from 6-phosphogluconate were found to be present in these cells. No 6-phosphogluconate dehydrogenase was detected. Oxidation of specifically labeled pyruvate gave the following (14)CO(2) evolution pattern: C-1=78%, C-2=48%, and C-3=37%; the pattern from acetate was C-1=73%; and C-2=56%. Oxidation of glutamate showed the preferential rate of (14)CO(2) evolution to be C-1 > C-2=C-5 > C-3, 4, whereas a higher yield of (14)CO(2) was obtained from the C-1 and C-4 carbons of succinate than from the C-2 and C-3 carbons. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the catabolic pathways of glucose oxidation in R. japonicum.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

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.