Hydrogenase in Frankia KB5: Expression of and relation to nitrogenase

2000 ◽  
Vol 46 (12) ◽  
pp. 1091-1095 ◽  
Author(s):  
Ulrika Mattsson ◽  
Anita Sellstedt
Keyword(s):  
Nitrogenase Activity ◽  
Large Subunit ◽  
Hydrogen Uptake ◽  
Hydrogenase Activity ◽  
Free Living ◽  
Monitoring Activity

The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.Key words: hydrogenase, expression, nitrogenase, Frankia KB5.

Effect of hydrogen and oxygen on uptake-hydrogenase activity in nitrogen-fixing and ammonium-grown Azospirillum brasilense

1983 ◽  
Vol 29 (9) ◽  
pp. 1119-1125 ◽  
Author(s):  
Karl H. Tibelius ◽  
Roger Knowles
Keyword(s):  
Azospirillum Brasilense ◽  
Nitrogenase Activity ◽  
Hydrogen Uptake ◽  
Rapid Decline ◽  
Hydrogenase Activity ◽  
Uptake Hydrogenase ◽  
Batch Cultures ◽  
Whole Cells ◽  
Irreversible Inhibition ◽  
Microaerobic Conditions

Hydrogen uptake by Azospirillum brasilense was studied in N2-fixing and NH4Cl-grown batch cultures. The Km for H2 of uptake hydrogenase (O2-dependent H3H uptake) in whole cells was 9 µM. In N2-flxing or NH4Cl-grown cultures hydrogenase activity reached its maximum in late log phase, followed by a rapid decline in stationary phase. In cultures sparged with H2, hydrogenase activity was markedly prolonged. Rates of O2-dependent H3H uptake and H2 uptake measured by gas chromatography were very similar. Hydrogenase derepression required microaerobic conditions, was independent of nitrogenase derepression, did not require exogenous H2, and may be enhanced by electron-donor limitation. Air caused irreversible inhibition of hydrogenase activity. In N-free cultures, theO2 optima for H2 uptake, ranging from 0.5 to 1.25% O2 depending on the phase of growth, were significantly higher than those for nitrogenase activity (C2H2 reduction), 0.15 to 0.35%, suggesting that H2 uptake may have a limited ability to aid in the protection of nitrogenase against inactivation by O2.

scholarly journals Engineering the Rhizobium leguminosarum bv. viciae Hydrogenase System for Expression in Free-Living Microaerobic Cells and Increased Symbiotic Hydrogenase Activity

2002 ◽  
Vol 68 (5) ◽  
pp. 2461-2467 ◽  
Author(s):  
B. Brito ◽  
J. M. Palacios ◽  
J. Imperial ◽  
T. Ruiz-Argüeso
Keyword(s):  
Time Course ◽  
Rhizobium Leguminosarum ◽  
Active Form ◽  
Hydrogen Uptake ◽  
Immunoblot Analysis ◽  
Living Cells ◽  
Culture Conditions ◽  
Hydrogenase Activity ◽  
Free Living ◽  
Cell Fractions

ABSTRACT Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.

scholarly journals Rhizobium leguminosarum hupE Encodes a Nickel Transporter Required for Hydrogenase Activity

2009 ◽  
Vol 192 (4) ◽  
pp. 925-935 ◽  
Author(s):  
Belén Brito ◽  
Rosa-Isabel Prieto ◽  
Ezequiel Cabrera ◽  
Marie-Andrée Mandrand-Berthelot ◽  
Juan Imperial ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Hydrogen Uptake ◽  
Site Directed Mutagenesis ◽  
Hydrogenase Activity ◽  
Rhizobium Tropici ◽  
Free Living ◽  
Mesorhizobium Loti ◽  
Nickel Uptake

ABSTRACT Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX5DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.

scholarly journals Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 952
Author(s):  
Paula Bellés-Sancho ◽  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Marta Adriana Pinto-Carbó ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Root Nodules ◽  
Growth Conditions ◽  
Lysine Biosynthesis ◽  
Metabolome Analysis ◽  
Plant Host ◽  
Free Living ◽  
Bacterial Enzyme ◽  
Homocitrate Synthase ◽  
Iron Molybdenum Cofactor

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

Use of products of straw decomposition by N2-fixing (C2H2-reducing) populations of bacteria in three soils from wheat-growing areas

1986 ◽  
Vol 37 (1) ◽  
pp. 1 ◽  
Author(s):  
MM Roper ◽  
DM Halsall
Keyword(s):  
Nitrogenase Activity ◽  
New South ◽  
Carbon Sources ◽  
Clay Content ◽  
Free Living ◽  
Straw Decomposition ◽  
South Wales ◽  
History Of ◽  
Straw Incorporation ◽  
The Difference

The potential for N2 fixation by free-living bacteria using straw as a source of energy was evaluated in three soils (one from Gunnedah and two from Cowra) representative of the wheat belt in New South Wales. All three soils had a history of straw incorporation. The abilities of the respective microbial populations to use a range of carbon sources, including potential products of decomposition of straw, was determined and compared with the size and composition of each population. Neutral to alkaline (pH 7.4) soil of high (51%) clay content from Gunnedah produced higher rates of nitrogenase activity with straw than more acid (pH 5.6) lower (17%) clay containing soil from Cowra (site B). Gunnedah soil also contained a larger population of N2-fixing bacteria which used a broader range of energy sources than soil from either Cowra site B or Cowra site W (pH 5.8, clay content 34%). There was little difference in the composition of the N2-fixing populations in each of the soils except that Azotobacter spp. were absent from the acid Cowra soils. It was concluded that the difference in behaviour of the respective N2-fixing populations was primarily due to the physical characteristics of the soil affecting the numbers and activities of diazotrophic microorganisms. In addition some soil environments failed to support specific organisms.

Hydrogen Evolution Catalyzed by Hydrogenase in Cultures of Cyanobacteria

1981 ◽  
Vol 36 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Patrick C. Hallenbeck ◽  
Leon V. Kochian ◽  
John R. Benemann
Keyword(s):  
Hydrogen Production ◽  
Hydrogen Evolution ◽  
High Frequency ◽  
Nitrogenase Activity ◽  
Inhibitory Effect ◽  
Hydrogenase Activity ◽  
Oxygen Inhibition ◽  
Oxygen Sensitive

Abstract Cultures of Anabaena cylindrica, grown on media containing 5 mᴍ NH4Cl (which represses heterocyst formation), evolved hydrogen after a period of dark incubation under an argon atmosphere. This hydrogen production was not due to nitrogenase activity, which was nearly undetectable, but was due to a hydrogenase. Cultures grown on media with tungsten substituted for molybdenum had a high frequency of heterocysts (15%) and inactive nitrogenase after nitrogen starvation. The hydrogenase activity of these cultures was three-fold greater than the activity of non-heterocystous cultures. The effects of oxygen inhibition on hydrogen evolution by hetero-cystous cultures suggest that two pools of hydrogenase activity exist - an oxygen sensitive hydrogen evolution in vegetative cells and a relatively oxygen-resistent hydrogen evolution in heterocysts. In either case, inhibition by oxygen was reversible. Light had an inhibitory effect on net hydrogen evolution. Hydrogen production in vitro was much higher than in vivo, indicating that in vivo hydrogenase activity is limited by endogenous reductant supply.

scholarly journals Paraburkholderia phymatum STM815 σ54 Controls Utilization of Dicarboxylates, Motility, and T6SS-b Expression

Nitrogen ◽  
2020 ◽  
Vol 1 (2) ◽  
pp. 81-98
Author(s):  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Samanta Bolzan de Campos ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Nitrogenase Activity ◽  
Secretion Systems ◽  
Free Living ◽  
Major Life ◽  
Life Styles ◽  
Type Vi Secretion ◽  
Flagellar Biosynthesis ◽  
Free Living Conditions

Rhizobia have two major life styles, one as free-living bacteria in the soil, and the other as bacteroids within the root/stem nodules of host legumes where they convert atmospheric nitrogen into ammonia. In the soil, rhizobia have to cope with changing and sometimes stressful environmental conditions, such as nitrogen limitation. In the beta-rhizobial strain Paraburkholderia phymatum STM815, the alternative sigma factor σ54 (or RpoN) has recently been shown to control nitrogenase activity during symbiosis with Phaseolus vulgaris. In this study, we determined P. phymatum’s σ54 regulon under nitrogen-limited free-living conditions. Among the genes significantly downregulated in the absence of σ54, we found a C4-dicarboxylate carrier protein (Bphy_0225), a flagellar biosynthesis cluster (Bphy_2926-64), and one of the two type VI secretion systems (T6SS-b) present in the P. phymatum STM815 genome (Bphy_5978-97). A defined σ54 mutant was unable to grow on C4 dicarboxylates as sole carbon source and was less motile compared to the wild-type strain. Both defects could be complemented by introducing rpoNin trans. Using promoter reporter gene fusions, we also confirmed that the expression of the T6SS-b cluster is regulated by σ54. Accordingly, we show that σ54 affects in vitro competitiveness of P. phymatum STM815 against Paraburkholderia diazotrophica.

Research on Improvement of Nitrogenase Activity of Free-Living Nitrogen-Fixing Bacteria Using DBD Plasma

2013 ◽  
Vol 671-674 ◽  
pp. 2674-2678 ◽  
Author(s):  
Yan Yun Zhu ◽  
Xiao Li Zhu ◽  
Fang She Yang
Keyword(s):  
Rhizosphere Soil ◽  
Barrier Discharge ◽  
Nitrogenase Activity ◽  
Nitrogen Fixing ◽  
Bacterial Strains ◽  
Dbd Plasma ◽  
Nitrogen Fixing Bacteria ◽  
Free Living ◽  
Reduction Assay ◽  
Rrna Sequencing

Nitrogen-fixing bacteria were screened from the rhizosphere soil of plants in Shaanxi in China. 36 free-living nitrogen-fixing bacterial strains were isolated and their nitrogenase activity were determined by acetylene reduction assay (ARA), two strains named FLNB03 and FLNB09 with higher nitrogenase activity were isolated and identified by 16S rRNA sequencing. The datum showed that FLNB03 was similar to Acinetobacter and their similarity reached 99%, FLNB09 was similar to Agrobacterium sp. and their similarity reached 99%. Then both of them were treated using Dielectric Barrier Discharge (DBD) plasma for mutation and their mutants called FLNB03-2 and FLNB09-3 were obtained. The nitrogenase activity of FLNB03-2 was 0.61±0.10 nmol•107cfu-1•h-1, and that of FLNB09-3 was 0.40±0.05 nmol•107cfu-1•h-1, their nitrogenase activity increased by 22.00% and 14.29% than their original bacteria respectively. FLNB03-2 and FLNB09-3 might be used as microbial fertilizer.

Field measurements of nitrogenase activity in soils amended with wheat straw

1983 ◽  
Vol 34 (6) ◽  
pp. 725 ◽  
Author(s):  
MM Roper
Keyword(s):  
Nitrogenase Activity ◽  
New South ◽  
Field Measurements ◽  
Field Capacity ◽  
Nitrogen Fertilizers ◽  
Free Living ◽  
Straw Decomposition ◽  
Indirect Measure ◽  
South Wales

Nitrogenase activity and decomposition of straw were examined in situ in two areas (Gunnedah and Cowra) representative of large areas of the New South Wales wheat belt. Measurements of nitrogenase activity were made by adapting the acetylene reduction assay for use in the field. Evolution of CO2 was monitored as an indirect measure of decomposition of straw. The addition of straw to soil stimulated nitrogenase activity which was related to the amount of straw added and the rate of straw decomposition. There were significant levels of activity provided the soil was moist and warm. Nitrogenase activity increased with mean daily soil temperature (up to at least 30�C) and decreased as the soil dried from field capacity. It is concluded that nitrogen fixation by free-living nitrogen-fixing microorganisms in soils amended with straw may contribute to the nitrogen status of the soil and thus reduce the need for nitrogen fertilizers.

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