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.     

The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti

Summary The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wildtype symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix⁺. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon; rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.     

Cloning of a DNA region from Bradyrhizobium japonicum encoding pleiotropic functions in heme metabolism and respiration

Random and site-directed Tn5-induced mutagenesis of Bradyrhizobium japonicum yielded two mutations, one in strain 2960 and the other in strain 2606::Tn5-20, which mapped close to each other but in separate genes. The corresponding wild-type genes were cloned, and their approximate location on the cloned DNA was determined. Mutant 2960 was Fix^- and formed green nodules on soybean, whereas strain 2606::Tn5-20 had ca. 4% of wild-type Fix activity and formed white nodules. Cytochrome oxidase assays (Nadi tests) showed a negative reaction with both mutants, indicating a functional deficiency of cytochrome c or its terminal oxidase or both. However, the mutants grew well under aerobic conditions on minimal media with different carbon sources. Furthermore, mutant 2960 had a reduced activity in hydrogen uptake, was unable to grow anaerobically with nitrate as the terminal electron acceptor and 2960-infected soybean nodules contained little, if any, functional leghemoglobin. Southern blot analysis showed that a B. japonicum heme biosynthesis mutant [strain LO505: O'Brian MR, Kirshbom PM, Maier RJ (1987) Proc Natl Acad Sci USA 84: 8390–8393] had its mutation close to the Tn5 insertion site of our mutant 2606::Tn5-20. This finding, combined with the observed phenotypes, suggested that the genes affected in mutants 2960 and 2606::Tn5-20 were involved in some steps of heme biosynthesis thus explaining the pleiotropic respiratory deficiencies of the mutants. Similar to strain LO505, the mutant 2606::Tn5-20 (but not 2960) was defective in the activity of protoporphyrinogen IX oxidase which catalyzes the penultimate step in the heme biosynthesis pathway. This suggests that one of the two cloned genes may code for this enzyme.     

Metabolomic profiling of wild‐type and mutant soybean root nodules using laser‐ablation electrospray ionization mass spectrometry reveals altered metabolism

The establishment of the nitrogen‐fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization‐mass spectrometry (LAESI‐MS) for in situ metabolic profiling of wild‐type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl‐acyl carrier protein desaturase (sacpd‐c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix⁺) and ineffective (nifH mutant, fix^?) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd‐c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI‐MS for high‐throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.     

An analysis of gene expression data involving examination of signaling pathways activation reveals new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia

Androgenetic alopecia is the most common form of hair loss. Minoxidil has been approved for the treatment of hair loss, however its mechanism of action is still not fully clarified. In this study, we aimed to elucidate the effects of 5% minoxidil topical foam on gene expression and activation of signaling pathways in vertex and frontal scalp of men with androgenetic alopecia. We identified regional variations in gene expression and perturbed signaling pathways using in silico Pathway Activation Network Decomposition Analysis (iPANDA) before and after treatment with minoxidil. Vertex and frontal scalp of patients showed a generally similar response to minoxidil. Both scalp regions showed upregulation of genes that encode keratin associated proteins and downregulation of ILK, Akt, and MAPK signaling pathways after minoxidil treatment. Our results provide new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia.     

Variation in seed packaging of a fleshy‐fruited conifer provides insights into the ecology and evolution of multi‐seeded fruits

• The study of intraspecific seed packaging (i.e. seed size/number strategy) variation across different populations may allow better understanding of the ecological forces that drive seed evolution in plants. Juniperus thurifera (Cupressaceae) provides a good model to study this due to the existence of two subspecies differentiated by phenotypic traits, such as seed size and cone seediness (number of seeds inside a cone), across its range.
• The aim of this study was to analyse seed packaging (seed mass and cone seediness) variation at different scales (subspecies, populations and individuals) and the relationship between cone and seed traits in European and African J. thurifera populations.
• After opening more than 5300 cones and measuring 3600 seeds, we found that seed packaging traits followed different patterns of variation. Large‐scale effects (region and population) significantly contributed to cone seediness variance, while most of the seed mass variance occurred within individuals. Seed packaging differed between the two sides of the Mediterranean Sea, with African cones bearing fewer but larger seeds than the European ones. However, no differences in seed mass were found between populations when taking into account cone seediness. Larger cones contained more pulp and seeds and displayed a larger variation in individual seed mass.
• We validated previous reports on the intraspecific differences in J. thurifera seed packaging, although both subspecies followed the same seed size/number trade‐off. The higher seediness and variation in seed mass found in larger cones reveals that the positive relationship between seed and cone sizes may not be straightforward.We hypothesise that the large variation of seed size found within cones and individuals in J. thurifera, but also in other fleshy‐fruited species, could represent a bet‐hedging strategy for dispersal.