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 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 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 Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3535-3542 ◽  
Author(s):  
Zhihong Xie ◽  
Yuetang Dou ◽  
Shuzheng Ping ◽  
Ming Chen ◽  
Guoying Wang ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Pseudomonas Stutzeri ◽  
Physical Interaction ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Free Living ◽  
Yeast Two Hybrid System ◽  
Living State ◽  
Microaerobic Conditions ◽  
Two Hybrid

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

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 FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Liu ◽  
Xue Bai ◽  
Yan Li ◽  
Haikun Zhang ◽  
Xiaoke Hu
Keyword(s):  
Signal Transduction ◽  
Host Plant ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Response Regulators ◽  
Free Living ◽  
Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

Expression of the nodulation and nitrogen fixation genes in Rhizobium meliloti during development

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 354-360 ◽  
Author(s):  
San Chiun Shen ◽  
Shui Ping Wang ◽  
Guan Qiao Yu ◽  
Jia Bi Zhu
Keyword(s):  
Nitrogen Fixation ◽  
Rhizobium Meliloti ◽  
Abnormal Development ◽  
Wild Type ◽  
Free Living ◽  
Nod Genes ◽  
Nodule Initiation ◽  
Fix Genes ◽  
Nitrogen Fixation Genes

Genes that specify nodulation (nod genes) are only active in the free-living rhizobia or in the nodule initiation state of rhizobia. As soon as the repression of nod genes occurs in the bacteroids of the nodule, nifA is induced, while ntrC is inactivated and thus the nifA-mediated nif/fix genes are turned on. Limitation of available oxygen brings about the induction of nifA, which reflects the actual status of nif/fix gene activities in symbiotic state of rhizobia. Oxygen thus appears to be a major symbiotic signal to the expression of bacteroid nif/fix genes. Mutation of nifA or shortage of nifA product in wild-type rhizobia caused by the inhibition of multicopy nifH/fixA promoters leads to an abnormal development of nodules and premature degradation of bacteroids in nodules.Key words: nitrogen fixation, nodulation, nif/fix regulation, nifA mutant.

scholarly journals High-Titer, Wild-Type Free Recombinant Adeno-Associated Virus Vector Production Using Intron-Containing Helper Plasmids

2000 ◽  
Vol 74 (24) ◽  
pp. 11456-11463 ◽  
Author(s):  
Lei Cao ◽  
Yuhong Liu ◽  
Matthew J. During ◽  
Weidong Xiao
Keyword(s):  
Gene Expression ◽  
High Titer ◽  
Infectious Clone ◽  
Wild Type ◽  
Helper Plasmid ◽  
Intron Insertion ◽  
Adeno Associated Virus ◽  
Vector Systems ◽  
Simplex Virus ◽  
High Level

ABSTRACT Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate therep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

scholarly journals From Intracellular Bacteria to Differentiated Bacteroids: Transcriptome and Metabolome Analysis inAeschynomeneNodules Using theBradyrhizobiumsp. Strain ORS285bclAMutant

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Florian Lamouche ◽  
Anaïs Chaumeret ◽  
Ibtissem Guefrachi ◽  
Quentin Barrière ◽  
Olivier Pierre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptome Analysis ◽  
Soil Bacteria ◽  
Regulation Of Gene Expression ◽  
Nitrogen Fertilizers ◽  
Peptide Transporter ◽  
Intracellular Bacteria ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Free Living

ABSTRACTSoil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells, where they differentiate into nitrogen-fixing bacteroids. Several legume lineages force their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process inBradyrhizobiumsp. strain ORS285, a symbiont ofAeschynomenespp. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells, but they cannot differentiate into enlarged polyploid and functional bacteroids. Thus, thebclAnodule bacteria constitute an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules ofAeschynomene afrasperaandAeschynomene indicainfected with the wild type or thebclAmutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied by a first transcriptome switch involving several hundred upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving fewer genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a nonsymbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.IMPORTANCELegume-rhizobium symbiosis is a major ecological process, fueling the biogeochemical nitrogen cycle with reduced nitrogen. It also represents a promising strategy to reduce the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs, where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis ofBradyrhizobiumwild-type andbclAmutant bacteria in culture and in symbiosis withAeschynomenehost plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.

scholarly journals Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

2007 ◽  
Vol 189 (16) ◽  
pp. 5850-5859 ◽  
Author(s):  
Pier-Luc Tremblay ◽  
Thomas Drepper ◽  
Bernd Masepohl ◽  
Patrick C. Hallenbeck
Keyword(s):  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Pii Proteins ◽  
High Level

ABSTRACT Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.

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