scholarly journals nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth

2016 ◽  
Vol 29 (10) ◽  
pp. 767-773 ◽  
Author(s):  
Jenjira Wongdee ◽  
Pongpan Songwattana ◽  
Nico Nouwen ◽  
Rujirek Noisangiam ◽  
Joel Fardoux ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Transcriptional Analysis ◽  
Main Role ◽  
Free Living ◽  
Living State ◽  
Bradyrhizobium Sp ◽  
Reporter Strains ◽  
Free Living Conditions

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

scholarly journals Azorhizobium caulinodans PIIand GlnK Proteins Control Nitrogen Fixation and Ammonia Assimilation

1999 ◽  
Vol 181 (8) ◽  
pp. 2655-2658 ◽  
Author(s):  
Nathalie Michel-Reydellet ◽  
P. Alexandre Kaminski
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Ammonia Assimilation ◽  
Ammonium Excretion ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Free Living ◽  
Living State ◽  
High Level

ABSTRACT We herein report that Azorhizobium caulinodansPII and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation. The disruption of both glnB andglnK resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state. PII and GlnK also controllednif gene expression because NifA activated nifHtranscription and nitrogenase activity was derepressed in glnB glnK double mutants, but not in wild-type bacteria, grown in the presence of ammonia.

scholarly journals Artificial activation of nif gene expression in nodule bacteria Ex Planta

2019 ◽  
Vol 17 (2) ◽  
pp. 35-42
Author(s):  
Andrey K. Baymiev ◽  
Roman S. Gumenko ◽  
Anastasiya A. Vladimirova ◽  
Ekaterina S. Akimova ◽  
Zilya R. Vershinina ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Nitrogen Fixing ◽  
Nodule Bacteria ◽  
Free Living ◽  
Living State ◽  
Artificial Activation ◽  
Nifa Gene

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters. Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state. Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation. Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

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.

scholarly journals Poly-β-Hydroxybutyrate Turnover inAzorhizobium caulinodans Is Required for Growth and AffectsnifA Expression

1998 ◽  
Vol 180 (19) ◽  
pp. 5070-5076 ◽  
Author(s):  
Karine Mandon ◽  
Nathalie Michel-Reydellet ◽  
Sergio Encarnación ◽  
P. Alexandre Kaminski ◽  
Alfonso Leija ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Constitutive Expression ◽  
Reducing Power ◽  
Azorhizobium Caulinodans ◽  
Free Living ◽  
Nucleotide Pools ◽  
Living State ◽  
Mutant Strains ◽  
Growth Properties ◽  
Phb Synthase

ABSTRACT Azorhizobium caulinodans is able to fix nitrogen in the free-living state and in symbiosis with the tropical legumeSesbania rostrata. The bacteria accumulate poly-β-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase, phbC, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif−), and affected in nucleotide pools and induced Fix− nodules devoid of bacteria. The Nif− phenotype was the consequence of the lack ofnifA transcription. Nitrogenase activity was partially restored to a phbC mutant by constitutive expression of thenifA gene. However, this constitutive nifAexpression had no effect on the nucleotide content or on growth of thephbC mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control ofnifA expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents.

scholarly journals Multiple groESL Operons Are Not Key Targets of RpoH1 and RpoH2 in Sinorhizobium meliloti

2006 ◽  
Vol 188 (10) ◽  
pp. 3507-3515 ◽  
Author(s):  
Alycia N. Bittner ◽  
Valerie Oke
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
High Temperature ◽  
Sigma Factor ◽  
Host Plants ◽  
Sinorhizobium Meliloti ◽  
Free Living ◽  
Genes Encoding ◽  
Multiple Copies ◽  
Free Living Conditions

ABSTRACT Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

scholarly journals Analysis of a Symbiosis-Specific Cytochrome P450 Homolog in Rhizobium sp. BR816

2001 ◽  
Vol 14 (7) ◽  
pp. 918-924 ◽  
Author(s):  
Ellen Luyten ◽  
Elfriede Swinnen ◽  
Katrien Vlassak ◽  
Christel Verreth ◽  
Bruno Dombrecht ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Transcriptional Analysis ◽  
Free Living ◽  
Amino Acid Similarity ◽  
Reading Frame ◽  
P450 Gene ◽  
Symbiotic Plasmid ◽  
Rhizobium Sp ◽  
Free Living Conditions

Sequence analysis of the DNA region upstream of nodO in Rhizobium sp. BR816 revealed an open reading frame in which the deduced amino acid sequence shows homology with cytochrome P450. Because the BR816 P450 homolog shows 73% amino acid similarity with CYP127A1(Y4vG), which is identified on the symbiotic plasmid of Rhizobium sp. NGR234, it is named CYP127A2. Transcriptional analysis of CYP127A2 revealed high expression in bacteroids, whereas no or hardly any expression was observed under free-living conditions. Low-level, free-living expression, however was noticed when cells were grown microoxically at acid pH levels. A number of possible substrates that may induce P450 gene expression were analyzed, but only the addition of short-chain alcohols to cultures slightly increased CYP127A2 expression. High levels of CYP127A2 expression observed in bacteroids of a nifH mutant strain, which formed non-fixing nodules on bean, indicated that the genuine substrate for CYP127A2 is not a metabolite resulting from N2-fixation. Nevertheless, expression analysis pointed to a NifA- and σ54-dependent transcription.

scholarly journals Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

2012 ◽  
Vol 78 (22) ◽  
pp. 8056-8061 ◽  
Author(s):  
Ji Xu ◽  
Xiao-Lin Li ◽  
Li Luo
Keyword(s):  
Nitrogen Fixation ◽  
Drought Stress ◽  
Sinorhizobium Meliloti ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Severe Drought ◽  
Control Strain ◽  
Free Living ◽  
Content Type ◽  
Host Tolerance

ABSTRACTCytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineeredSinorhizobiumstrains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed theAgrobacterium iptgene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although theiptgene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineeredSinorhizobiumstrains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.

scholarly journals Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

1999 ◽  
Vol 12 (11) ◽  
pp. 1008-1015 ◽  
Author(s):  
Mario Ramírez ◽  
Brenda Valderrama ◽  
Raúl Arredondo-Peter ◽  
Mario Soberón ◽  
Jaime Mora ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Heterologous Expression ◽  
Energy Content ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rhizobium Etli ◽  
Free Living ◽  
Cell Extracts ◽  
Bean Plants

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

scholarly journals Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

2009 ◽  
Vol 191 (8) ◽  
pp. 2593-2600 ◽  
Author(s):  
Chrysanthi Kalloniati ◽  
Daniela Tsikou ◽  
Vasiliki Lampiri ◽  
Mariangela N. Fotelli ◽  
Heinz Rennenberg ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Carbonic Anhydrase ◽  
Mutant Strain ◽  
Symbiotic Nitrogen Fixation ◽  
Plant Traits ◽  
Nitrogenase Activity ◽  
Nodule Development ◽  
Free Living ◽  
Model Legume ◽  
Mesorhizobium Loti

ABSTRACT Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3 − produced by CAA1.

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