A pellet method to demonstrate nitrogen fixation by free-living rhizobia

Summary A method is described to demonstrate nitrogen fixation by free-living Rhizobium cells. After aerobic growth in a nutrient solution, the bacteria are centrifuged. Acetylene reduction by the rhizobial cells in the pellet can be measured within a few days. Hydrogen gas frequently stimulates acetylene reduction.     

Rhizobial polysaccharide-degrading enzymes from roots of legumes

The surface structure and chemistry of symbiotic bacteria from the genus Rhizobium are probably important for the outcome of the infection of legume hosts. Exopolysaccharide, capsular polysaccharide, lipopolysaccharide and a low-molecular-weight polysaccharide were isolated from R. trifolii UTC 110-1 and R. leguminosarum UTC 114-5 and partially characterized. No or only minor differences in sugar composition could be found for the corresponding fractions from the two organisms. A general method to measure low activities of polymer-degrading enzymes was developed, and used to determine enzyme activities in root extracts of Trifolium repens L. cv. Lena and Pisum xativiini L. cv. Little Marvel against the isolated rhizobial polysaccharides. An enzyme preparation from T. repens partially degraded all polysaccharides isolated from its symbiont R. trifolii while polysaccharides from R. leguminosarum , symbiont of P. sativum , were degraded to a much lesser extent. Correspondingly, an enzyme preparation from P. sativum degraded all polysaccharides isolated from both its symbiont R. leguminosarum and its non-symbiont R. trifolii. The amount of symbiont polysaccharides degraded was larger than the amount of polysaccharides degraded from the non-symbiont R. trifolii.     

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.     

Alleviating the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticum – fenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment

Rhizobia-legume symbiosis depends on molecular dialog, which involves the production of specific plant flavonoid compounds as signal molecules. Rhizobium tibeticum was recovered from the root nodule of fenugreek and identified by sequencing the 16S rRNA gene. The effect of salinity stress on nod gene expression was measured in terms of β-galactosidase activity. R. tibeticum containing Escherichia coli lacZ gene fusions to specific nodulation (nod) genes were used to determine β-galactosidase activity. Combination of hesperetin (7.5 µM) and apigenin (7.5 µM) significantly increased β-galactosidase activity more than the single application of hesperetin or apigenin. Preincubation of R. tibeticum with hesperetin and apigenin combination significantly alleviates the adverse effect of salinity on nod gene expression and therefore, enhances nodulation and nitrogen fixation of fenugreek.     

Decoupled genomic elements and the evolution of partner quality in nitrogen‐fixing rhizobia

Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long‐term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long‐term nitrogen fertilization caused the evolution of less‐mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less‐mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long‐term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.     

黄芪多糖对咪喹莫特诱导小鼠银屑病样皮炎的干预作用及其机制

目的:探讨黄芪多糖对咪喹莫特诱导的小鼠银屑病样皮炎的干预作用及其机制。方法:40只健康雌性C57BL/6小鼠随机分为空白对照组、模型组、黄芪多糖高剂量组(200 mg/kg)、中剂量组(100 mg/kg)和低剂量组(50 mg/kg),共5组,每组8只,利用5%咪喹莫特乳膏涂抹小鼠背部制备银屑病样皮炎模型,监测各组小鼠PASI评分,并采用ELISA测定小鼠炎症因子分泌的变化,通过流式细胞术检测皮炎中巨噬细胞的浸润程度。结果:与空白对照组比较,模型组小鼠PASI评分明显增加(P<0.05),血清TNF-α、IL-1β和IL-6水平明显升高(P<0.05),皮肤组织中浸润的巨噬细胞明显增加(P<0.05);与模型组比较,黄芪多糖高、中剂量组小鼠PASI评分明显明显减少(P<0.05),小鼠血清TNF-α、IL-1β和IL-6水平明显下调(P<0.05);黄芪多糖高剂量组小鼠皮肤组织中浸润的巨噬细胞明显减少(P<0.05)。结论:黄芪多糖通过抑制皮肤组织中巨噬细胞浸润,降低血清中TNF-α、IL-1β和IL-6的分泌,来改善小鼠银屑病样皮炎。     

Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Abstract Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.     

Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

Aims: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro‐ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. Methods and Results: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal‐nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. Conclusions: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal‐nodulating rhizobia could enhance plant growth. Significance and Impact of the Study: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.