Carbon Metabolism Enzymes of Rhizobium meliloti Cultures and Bacteroids and Their Distribution within Alfalfa Nodules

Several carbon metabolism enzymes were measured in cultured cells and bacteroids of Rhizobium meliloti 102F51 and in alfalfa root nodule cytosol. The enzyme activity levels of the pentose phosphate pathway were much higher than those of the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways in extracts of cultured cells. The pattern of enzyme activities in the bacteroids was different from that of cultured cells.     

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.

A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.     

Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids: purification and properties

Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids has been purified greater than 130-fold. The enzyme has a molecular weight of 180,000 +/- 15,000 and consists of four identical subunits of 44,000 +/- 2,000. The enzyme was specific for acetoacetyl-CoA; ketodecanoyl-CoA did not serve as a substrate. Catalysis proceeds via a ping-pong mechanism. Iodoacetamide effectively inhibited the enzyme but acetoacetyl-CoA provided considerable protection against this compound. Magnesium was found to inhibit both the thiolysis reaction and the condensation reaction. Acetoacetyl-CoA thiolysis activity was not affected by potassium, ammonium, or several organic acids but was found to be inhibited by NADH. The inhibition by NADH may have an effect during the decline of the symbiosis.     

A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum

Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2-D PAGE using narrow range IPG strips. More than 1200 proteins were detected, of which nearly 500 proteins were analysed by MALDI-TOF and 310 spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free-living B. japonicum. A comparative analysis of proteomes of free-living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states.     

Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC₅₀ values ranging form 0.3–1 μM for compound 42, and 0.1–0.3 μM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.