Light microscopy of early stages in the symbiosis of soybean with a delayed-nodulation mutant of Bradyrhizobium japonicum

An -ketoglutarate dehydrogenase mutant (LSG184) of Bradyrhizobium japonicum USDA110 has a delayed nodulation phenotype when inoculated onto soybean (Glycine max L.). To pinpoint the defective stage of symbiotic development, light microscopic techniques were used to monitor early responses of soybean to inoculation with the mutant as compared to the wildtype strain. Methylene blue was used to visualize curled root hairs and a convenient haematoxylin staining method was developed that could detect nodule primordia as early as 2 d after inoculation. The results demonstrate that early symbiotic events occur with normal timing after inoculation with SLG184 and that its developmental delay is first evident during the progression of nodule primordia into emergent nodules. The timing of this delay suggests that LSG184 is not deficient in Nod factor production, at least during the early stages of symbiosis, but rather may have a defect in infection thread initiation or elongation. The results further imply that the rate of development of advanced soybean nodule primordia is, in part, dependent on the metabolic capabilities of the invading bacterium.     

Phosphoenolpynivate carboxylase, malate and alcohol dehydrogenase activities in alfalfa (Medicago sativa) nodules under water stress

The effect of drought upon phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), malate ddiydrogenase (MDH; EC 1.1.1.37), alcohol dehydrogenase (ADH; EC 1.1.1.1) and β -hydroxybulyrate dehydrogenase ( β -OH-BDH; EC 1.1.1.30) enzyme activities as well as the leghemoglobin (Lb), malate and ethanol contents of alfalfa nodules ( Medicago sativa L. cv. Aragon) were examined. Both the ieghemoglobin (Lb) content and the Lb/soluble protein ratio were significantly reduced at a nodule water potential (Ψ_nod) of—1.3 MPa. At lower Ψ_nod, Lb content decreased further, but the ratio remained unchanged. Slight stress (—1.3 MPa) drastically affected acetylene reduction activity (ARA; 60% reduction) whereas in vitro PEPC activity was main-tained at relatively constant values. As stress progressed (—2.0 MPa), a simultaneous reduction in both activities was observed. Severe stress (Ψ_nod lower than —2.0 MPa) stimulated in vitro PEPC. Bacteroid β -J-OH-BDH activity was stimulated by slight (—1.3 MPa) and moderate (—2.0 MPa) drought. MDH activity rose in slightly stressed nodules (Ψ_nod—1.3 MPa). Greater water deficits sharply decreased MDH activity to values significantly lower than those found in control nodules. Nodule malate content followed the same pattern as MDH. The plant fraction of the nodule showed constitutive ADH activity and contained ethanol. ADH was stimulated at slight (— 1.3 MPa) and moderate drought levels (—2.0 MPa). Ethanol content showed similar behavior to ADH activity. Inhibition of ARA, reduction of Lb content and stimulation of the fermentative metabolism induced by water stress suggest some reduction ira O₂ availability within the nodule.     

The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum

A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Uniformity of the microsymbiont population from soybean nodules with respect to buoyant density

The microsymbiont population in soybean root nodules (Glycine max L. cv Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development to determine the extent of heterogeneity in this population. The microsymbiont population was isolated by centrifugation through a continuous sucrose gradient (44 to 57% weight to weight ratio) and appeared homogeneous at each age examined up to 26 days after planting based on the symmetrical distribution of the population, enzyme activities, poly-β-hydroxybutyrate contents, protein contents, and viabilities. Some differences in viability, protein content, and acetylene reduction activity were observed at later ages. The population migrated to progressively lighter buoyant densities with increasing age until a density equivalent to 48% sucrose was reached. The changing density correlated directly with the increasing poly-β-hydroxybutyrate to protein ratio. The acetylene reduction activity, based on microsymbiont concentration, followed the same developmental pattern as whole nodules. On a protein basis, the decline of acetylene reduction activity was later and reflected the decrease in protein content per cell. These results suggested that the microsymbiont population, which resulted from inoculation of B. japonicum 2143 onto Williams 82 cultivar of soybeans, developed as a homogeneous population.     

Adenylate cyclase and cyclic AMP phosphodiesterase in Bradyrhizobium japonicum bacteroids.

Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.     

Enzymes of the Poly-beta-Hydroxybutyrate and Citric Acid Cycles of Rhizobium japonicum Bacteroids

The activities of several enzymes of the citric acid and poly-β-hydroxybutyrate cycles were measured in Rhizobium japonicum 3I1B-143 bacteroids which had been isolated from soybean nodules by sucrose gradient centrifugation. During the period of developing nitrogenase activity, the specific activity of fumarase, hydroxybutyrate dehydrogenase, β-ketothiolase, and pyruvate dehydrogenase complex increased whereas acetoacetate-succinyl-CoA transferase and isocitrate dehydrogenase decreased. Malate dehydrogenase activity remained constant. The amount of available acetyl-CoA, based on pyruvate dehydrogenase activity, should be sufficient to support both metabolic cycles concurrently. The temporal relationship between nitrogenase activity and poly-β-hydroxybutyrate accumulation has been reexamined.     

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Hydrogen-Dependent Nitrogenase Activity and ATP Formation in Rhizobium japonicum Bacteroids

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H₂-oxidizing system that recycles all of the H₂ lost through nitrogenase-dependent H₂ evolution. The addition of 72 μM H₂ to suspensions of bacteroids increased O₂ uptake 300% and the rate of C₂H₂ reduction 300 to 500%. The optimal partial pressure of O₂ was increased, and the partial pressure of O₂ range for C₂H₂ reduction was extended by adding H₂. A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H₂. Both H₂ and succinate provided respiratory protection for the N₂-fixing system in bacteroids. The oxidation of H₂ by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H₂ oxidation, the addition of H₂ increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O₂ indicated that the H₂-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H₂-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H₂ to H₂ uptake-negative strains of R. japonicum had no effect on ATP formation or C₂H₂ reduction. It is concluded that the H₂-oxidizing system in H₂ uptake-positive bacteroids benefits the N₂-fixing process by providing respiratory protection of the O₂-labile nitrogenase proteins and generating ATP to support maximal rates of C₂H₂ reduction by oxidation of the H₂ produced from the nitrogenase system.     

Nanotechnology as an adjunct tool for transplanting engineered cells and tissues

Laboratory and clinical studies have provided evidence of feasibility, safety and efficacy of cell transplantation to treat a wide variety of diseases characterized by tissue and cell dysfunction ranging from diabetes to spinal cord injury. However, major hurdles remain and limit pursuing large clinical trials, including the availability of a universal cell source that can be differentiated into specific cellular phenotypes, methods to protect the transplanted allogeneic or xenogeneic cells from rejection by the host immune system, techniques to enhance cellular integration of the transplant within the host tissue, strategies for in vivo detection and monitoring of the cellular implants, and new techniques to deliver genes to cells without eliciting a host immune response. Finding ways to circumvent these obstacles will benefit considerably from being able to understand, visualize, and control cellular interactions at a sub-micron level. Cutting-edge discoveries in the multidisciplinary field of nanotechnology have provided us a platform to manipulate materials, tissues, cells, and DNA at the level of and within the individual cell. Clearly, the scientific innovations achieved with nanotechnology are a welcome strategy for enhancing the generally encouraging results already achieved in cell transplantation. This review article discusses recent progress in the field of nanotechnology as a tool for tissue engineering, gene therapy, cell immunoisolation, and cell imaging, highlighting its direct applications in cell transplantation therapy.