Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.     

Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.     

Bradyrhizobium japonicum mutants defective in nitrogen fixation and molybdenum metabolism.

Bradyrhizobium japonicum JH mutants deficient in molybdenum metabolism into the enzymes nitrogenase and nitrate reductase were isolated by using the vector pSUP1011, which carries transposon Tn5 (streptomycin and kanamycin resistance). Mutants in Mo metabolism were obtained at a frequency of 3.6 X 10(-3) (per Kan Strr colony). The mutants were detected by their poor ability to grow in nitrate-containing medium without added Mo. One of the mutant types required 10(5) times more molybdate than the wild type to obtain maximal nitrogen fixation activity. Double-reciprocal plots of Mo uptake versus concentration indicated that the wild-type strain had a high- and a lower-affinity component for Mo binding. Mutant strains JH-90 and JH-119 lacked the high-affinity Mo uptake component and were also clearly deficient in Mo accumulation into a nonexchangeable form. Nitrogenase activity as well as Mo uptake ability could be restored in strains JH-90 and JH-119 by the addition of the sterile supernatant fraction of the wild type. Therefore, mutant strains JH-90 and JH-119 appeared to be deficient in an extracellular Mo-binding factor produced by the wild type. Mutant strains JH-14 and JH-143 had Mo uptake kinetics like those of the wild type (both high- and low-affinity binding for Mo) and appeared to be deficient in intracellular Mo metabolism processes. The addition of the wild-type supernatant did not restore Mo uptake or nitrogenase activity in these strains.     

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).     

Control analysis of photosynthetic CO2 fixation

The potential of control analysis to aid our understanding of regulation and control of photosynthetic carbon metabolism is investigated. Methods of metabolic control analysis are used to determine flux control coefficients of photosynthetic reactions from enzyme elasticities. Equations expressing control coefficients symbolically by enzyme elasticities are derived, and general properties of these expressions are analysed. Suggestions for experimental determination of flux control coefficients from enzyme elasticities are given. A simplified model of the Calvin-Benson cycle is used to illustrate interrelations between patterns of photosynthetic metabolites and that of control coefficients.Abbreviations GAPDH glyceraldehyde phosphate dehydrogenase - PGA 3-phosphoglycerate - PGK 3-phosphoglycerate kinase - Pi inorganic phosphate - PRK phosphoribulokinase - RuBP ribulose-1,5-bisphosphate(_{total, free}) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose-5-phosphate     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Citrate as a siderophore in Bradyrhizobium japonicum.

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.     

Effects of temperature and CO2 concentration on photosynthetic CO2 fixation by Chlorella

The rates of photosynthetic ¹⁴CO₂ fixation by Chlorella vulgarisllh, grown under high CO₂, were determined between 4 to 37°Cwith air containing from 300 to 13,000 ppm ¹⁴CO₂. When the CO₂level was increased, both the rate of photosynthesis and theoptimum temperature for maximum photosynthesis increased. Themaximum photosynthetic rate was reached at 12°C with 300ppm ^l4CO₂. Among the photosynthetic products fromed at 300 ppm ¹⁴CO₂, glycolatedecreased greatly when the temperature was raised from 20 to30°C. At 3,000 ppm ¹⁴CO₂ an insignificant amount of glycolatewas formed at all temperatures, whereas ¹⁴C-incorporation intothe insoluble fraction, sucrose, and the lipid fraction wassignificantly higher than at 300 ppm ¹⁴CO₂. The ¹⁴C in sucrosewas greatly increased and the radioactivity in the insolublefraction decreased when the temperature was raised from 28 to36°C. (Received April 8, 1980; )     

Enhanced competitiveness of a Bradyrhizobium japonicum mutant strain improved for nodulation and nitrogen fixation

Bradyrhizobium japonicum strain TA-11NOD⁺, with altered indole biosynthesis, exhibited enhanced nodulation and nitrogen fixation on soybean in previous greenhouse studies. In this study, field experiments were conducted at Upper Marlboro, Maryland, in the summers of 1988 and 1993. In 1988, the site used was essentially free of soybean-nodulating bacteria and seed yield in plots inoculated with either I-110ARS or TA-11NOD⁺ was significantly higher by 12 or 20%, respectively, than that of the uninoculated controls. The 1993 site had an indigenous soil population (about 10⁴ cells g^-1) of symbiotically ineffective soybean-nodulating bacteria. Nevertheless, six-week-old Morgan soybean plants inoculated with strain TA-11NOD⁺ had 44% more nodules and exhibited 50% more nitrogen fixation by acetylene reduction when compared with plants that received the parental strain I-110ARS. Nodule occupancy, as determined using genetic markers for rifampicin and streptomycin resistance, was significantly higher for strain TA-11NOD⁺ than for strain I-110ARS. Overall, for the two years and the two soybean genotypes, the yield obtained with TA-11NOD⁺ was 6% higher than that obtained with I-110ARS. Competition experiments were conducted in the greenhouse and strain TA-11NOD⁺ was significantly more competitive than strain I-110ARS in competition with strains USDA 6 or USDA 438.     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )