Free-living Rhizobium Japonicum Research Articles

Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.

Nickel is a component of hydrogenase in Rhizobium japonicum.

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.

Hydrogen oxidation activity in membranes from Rhizobium japonicum

Membranes capable of oxidizing H 2 with O 2 as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H 2-uptake specific activities when isolated in the presence of an H 2 atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O 2-dependent H 2-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H 2-uptake activity was sedimented. The particulate and soluble fractions containing H 2-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H 2 uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H 2 uptake with O 2 as acceptor in membranes was approx. 7.2. H 2-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H 2-reduced minus O 2-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H 2-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H 2-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H 2-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.

Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum.

Membranes from free-living Rhizobium japonicum were isolated to study electron transport components involved in H2 oxidation. The H2/O2 uptake rate ratio in membranes was approximately 2. The electron transport inhibitors antimycin A, cyanide, azide, hydroxylamine, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited H2 uptake and H2-dependent O2 uptake significantly. H2-reduced minus O2-oxidized absorption difference spectra revealed peaks at 551.5, 560, and 603 nm, indicating the involvement of cytochromes c, b, and a-a3, respectively. H2-dependent cytochrome reduction was completely inhibited in the presence of 0.15 mM HQNO. This inhibition was relieved by the addition of 0.1 mM menadione. Evidence is presented for the involvement of two b-type cytochromes in H2 oxidation. One b-type cytochrome was not reduced by ascorbate and had an absorption peak at 560 nm. The reduction of this cytochrome by H2 was not inhibited by cyanide. A second b-type cytochrome, cytochrome b', was not reduced by H2 in the presence of cyanide. This cytochrome had an absorption peak at 558 nm. Carbon monoxide difference spectra with H2 as reductant provided evidence for the involvement of cytochrome o as well as cytochrome a3 in H2 oxidation. H2 uptake activity in cell-free extracts was inhibited by UV light irradiation. Most of the activity of the UV-treated extracts was restored with the addition of ubiquinone. The restored activity was inhibited by cyanide. A branched electron transport pathway from H2 to O2 is proposed.

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10-30%, glutamate dehydrogenase to 50-70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120-200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 μmol per g nodule fresh weight, as compared to 186 μmol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly-β-hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.

Organic acids and prolonged nitrogenase activity by non-growing, free-living Rhizobium japonicum

Rhizobium japonicum 61-A-101 grew and fixed nitrogen more effectively on media containing an organic acid and a pentose sugar than on media containing only one of these carbon sources. Peak specific activities in the range 10–15 nmol C2H4 · h-1 · mg protein-1 were found for these organisms in a spot of growth about 1 cm diameter on agar surfaces exposed to air. Increasing concentrations of the organic acids (succinate or malonate) in a medium containing arabinose resulted in longer lasting activity. The inclusion of a third carbon source, glycerol, gave activity which remained at the maximum from about the 8 to the 18 day after inoculation although no growth of the bacteria occurs during the last 8 or 10 days. At low concentration of organic acid l-arabinose was a much better carbon source for supporting nitrogenase activity of these organisms that the d-form. Both organic acids affected the morphology of the bacteria. Higher concentrations, especially of malonate, gave swollen and distorted cells. When bacteria growing on organic acid-containing agar plates were suspended and plated after appropriate dilution on yeast extract — mannitolglycerol agar there was heterogeneity of colony form, with up to 90% microcolonies after growth on high malonate concentrations. The effects of malonate may be correlated with characteristics of the bacteroid form inside the nodule which contains relatively high concentrations of organic acids, especially malonate.

Flow-microfluorometric analysis of bacteroids fractionated from alfalfa and soybean nodules by buoyant density sedimentation

The cell size and DNA and protein content of bacteroids fractionated from alfalfa and soybean nodules by buoyant density sedimentation were examined by laser flow-microfluorometry. In alfalfa nodules, these parameters were inversely related to bacteroid density. The bacteroids which sedimented at the highest density ( p = 1.268) resembled free-living Rhizobium meliloti in cell size and protein content but averaged slightly higher in DNA content. The bacteroids which sedimented at the lowest density ( p = 1.224) averaged larger in size and contained significantly more DNA and protein then free-living R. meliloti. Despite the difference in density, bacteroid fractions from soybean nodules and free-living Rhizobium japonicum were similar in all measured parameters.

The Occurrence and Role of Ubiquinone in Electron Transport to Oxygen and Nitrate in Aerobically, Anaerobically and Symbiotically Grown Rhizobium japonicum

SUMMARY: Ubiquinone was extracted from free-living Rhizobium japonicum, grown aerobically or anaerobically, and from the symbiotic bacteroid form; it was tentatively identified as the Q-10 homologue. The ubiquinone concentration was highest in symbiotically grown R. japonicum but the ratio ubiquinone:total cytochrome was about 1·5:1 in membrane particles from organisms grown under all three conditions. The ubiquinone was reduced 75% by NADH, completely oxidized by oxygen but not oxidized by nitrate. NADH oxidase activity and nitrate reductase activity in membrane particles from organisms grown under the different conditions were similar except that nitrate reductase activity was low in aerobically grown organisms. It is concluded that ubiquinone functions in electron transport to oxygen but not to nitrate.

Physiological Levels and Properties of Polynucleotide Phosphorylase of Rhizobium meliloti

Summary: Polynucleotide phosphorylase activity was demonstrated in free-living and the symbiotic bacteroid forms of Rhizobium meliloti and in free-living Rhizobium japonicum. Crude extracts of R. meliloti f-28 catalysed the polymerization of 3.0 μmol ADP (mg protein)-1 h-1. The polynucleotide phosphorylase activity of symbiotic R. meliloti was about 50% of that of free-living R. meliloti and remained almost constant throughout the development of the nodules. Partially-purified fractions of free-living R. meliloti f-28 catalysed the polymerization of ribonucleoside diphosphates, the phosphorolysis of synthetic homopolymers and natural heteropolymers, and the β-phosphate exchange reaction. The enzyme had a pH optimum of 8.0 and had an obligatory requirement for Mg2+ for maximum activity. The phosphorylase was not highly primer-dependent, had low specificity for guanosine-containing substrates, and exhibited cooperative-type kinetics.

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the MoFe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-section of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified MoFe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the MoFe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in a 270-fold enhancement of activity indicating the presence of active MoFe protein in these extracts.Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the MoFe component of nitrogenase is carried by Rhizobium.