Rhizobium etli Mutant Modulates Carbon and Nitrogen Metabolism in Phaseolus vulgaris Nodules

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared with CE3. However, plants inoculated with strain CE3-vhb, which express the Vitreoscilla sp. hemoglobin and also displays increased respiratory capacity, did not have altered ureide transport in N2-fixing plants. The data suggest that inoculation with special selected mutant strains of R. etli can modulate nodule N assimilation and N transport compounds.

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Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism

Molecular and Cellular Biology ◽

10.1128/mcb.1.2.158-164.1981 ◽

1981 ◽

Vol 1 (2) ◽

pp. 158-164

Author(s):

N S Dunn-Coleman ◽

E A Robey ◽

A B Tomsett ◽

R H Garrett

Keyword(s):

Neurospora Crassa ◽

Glutamate Dehydrogenase ◽

Nitrogen Metabolism ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Glutamate Synthase ◽

Wild Type ◽

Mutant Strains ◽

Synthase Regulation ◽

Glutamate Biosynthesis ◽

Glutamine Limitation

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

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Carbon and nitrogen metabolism in legume root nodules

Phytochemistry ◽

10.1016/0031-9422(80)83181-5 ◽

1980 ◽

Vol 19 (3) ◽

pp. 341-355 ◽

Cited By ~ 114

Author(s):

Stephen Rawsthorne ◽

Frank R. Minchin ◽

Rodney J. Summerfield ◽

Claire Cookson ◽

James Coombs

Keyword(s):

Nitrogen Metabolism ◽

Root Nodules ◽

Carbon And Nitrogen ◽

Carbon And Nitrogen Metabolism

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Inhibition of Glutamine Synthetase by Phosphinothricin Leads to Transcriptome Reprograming in Root Nodules of Medicago truncatula

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-11-0322 ◽

2012 ◽

Vol 25 (7) ◽

pp. 976-992 ◽

Cited By ~ 19

Author(s):

Ana R. Seabra ◽

Patrícia A. Pereira ◽

Jörg D. Becker ◽

Helena G. Carvalho

Keyword(s):

Glutamine Synthetase ◽

Medicago Truncatula ◽

Root Nodule ◽

Higher Plants ◽

Cell Metabolism ◽

Root Nodules ◽

Organic N ◽

N Availability ◽

Time Points ◽

N Assimilation

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

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Malic Enzyme Cofactor and Domain Requirements for Symbiotic N2 Fixation by Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.01425-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 160-168 ◽

Cited By ~ 11

Author(s):

Michael J. Mitsch ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Amino Acid ◽

Malic Enzyme ◽

Sinorhizobium Meliloti ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

High Ratio ◽

Terminal Region ◽

Wild Type ◽

Mutant Strains ◽

Alfalfa Root

ABSTRACT The NAD+-dependent malic enzyme (DME) and the NADP+-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N2 (Fix−) in alfalfa root nodules, whereas tme mutants are unimpaired in their N2-fixing ability (Fix+). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N2 fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N2 fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD+-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N2 fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H+ to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N2-fixing bacteroids.

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Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Molecular and Cellular Biology ◽

10.1128/mcb.1.2.158 ◽

1981 ◽

Vol 1 (2) ◽

pp. 158-164 ◽

Cited By ~ 15

Author(s):

N S Dunn-Coleman ◽

E A Robey ◽

A B Tomsett ◽

R H Garrett

Keyword(s):

Neurospora Crassa ◽

Glutamate Dehydrogenase ◽

Nitrogen Metabolism ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Glutamate Synthase ◽

Wild Type ◽

Mutant Strains ◽

Synthase Regulation ◽

Glutamate Biosynthesis ◽

Glutamine Limitation

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The Stringent Response Is Required for Amino Acid and Nitrate Utilization, Nod Factor Regulation, Nodulation, and Nitrogen Fixation in Rhizobium etli

Journal of Bacteriology ◽

10.1128/jb.187.15.5075-5083.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5075-5083 ◽

Cited By ~ 23

Author(s):

Arturo Calderón-Flores ◽

Gisela Du Pont ◽

Alejandro Huerta-Saquero ◽

Horacio Merchant-Larios ◽

Luis Servín-González ◽

...

Keyword(s):

Amino Acids ◽

Type Strain ◽

Wild Type Strain ◽

Stringent Response ◽

Insertion Mutant ◽

Rhizobium Etli ◽

Wild Type ◽

Nod Factor ◽

Truncated Protein ◽

Carbon And Nitrogen

ABSTRACT A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a ΔrelA ΔspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Ω element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-β-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

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A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Applied and Environmental Microbiology ◽

10.1128/aem.64.10.3954-3960.1998 ◽

1998 ◽

Vol 64 (10) ◽

pp. 3954-3960 ◽

Cited By ~ 42

Author(s):

Donald A. Phillips ◽

Eve S. Sande ◽

J. A. C. Vriezen ◽

Frans J. de Bruijn ◽

Daniel Le Rudulier ◽

...

Keyword(s):

Salt Stress ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Genetic Locus ◽

Wild Type ◽

Single Strain ◽

Reading Frame ◽

Carbon And Nitrogen ◽

Promoter Probe ◽

Inducible Genes

ABSTRACT Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [14C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [14C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxABfusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.

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A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

Applied and Environmental Microbiology ◽

10.1128/aem.03314-13 ◽

2014 ◽

Vol 80 (7) ◽

pp. 2094-2101 ◽

Cited By ~ 9

Author(s):

Marion Koch ◽

Nathanaël Delmotte ◽

Christian H. Ahrens ◽

Ulrich Omasits ◽

Kathrin Schneider ◽

...

Keyword(s):

Bradyrhizobium Japonicum ◽

Host Plants ◽

Root Nodule ◽

Coenzyme A ◽

Root Nodules ◽

Proteome Analysis ◽

Wild Type ◽

Content Type ◽

Mutant Cells ◽

Respective Host

ABSTRACTRhizobia have a versatile catabolism that allows them to compete successfully with other microorganisms for nutrients in the soil and in the rhizosphere of their respective host plants. In this study,Bradyrhizobium japonicumUSDA 110 was found to be able to utilize oxalate as the sole carbon source. A proteome analysis of cells grown in minimal medium containing arabinose suggested that oxalate oxidation extends the arabinose degradation branch via glycolaldehyde. A mutant of the key pathway genesoxc(for oxalyl-coenzyme A decarboxylase) andfrc(for formyl-coenzyme A transferase) was constructed and shown to be (i) impaired in growth on arabinose and (ii) unable to grow on oxalate. Oxalate was detected in roots and, at elevated levels, in root nodules of four differentB. japonicumhost plants. Mixed-inoculation experiments with wild-type andoxc-frcmutant cells revealed that oxalotrophy might be a beneficial trait ofB. japonicumat some stage during legume root nodule colonization.

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Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Planta ◽

10.1007/bf00392355 ◽

1986 ◽

Vol 168 (3) ◽

pp. 316-323 ◽

Cited By ~ 84

Author(s):

A. C. Kendall ◽

R. M. Wallsgrove ◽

N. P. Hall ◽

J. C. Turner ◽

P. J. Lea

Keyword(s):

Hordeum Vulgare ◽

Nitrogen Metabolism ◽

Glutamate Synthase ◽

Hordeum Vulgare L ◽

Carbon And Nitrogen ◽

Carbon And Nitrogen Metabolism

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Regulation of Phosphate Assimilation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽

10.1093/genetics/148.4.1689 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1689-1700 ◽

Cited By ~ 5

Author(s):

Sylvie D Bardin ◽

Turlough M Finan

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Phosphate Transport ◽

Wild Type ◽

Pi Transport ◽

Regulatory Systems ◽

Mutant Strains ◽

Insertion Mutations

Abstract We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF) encoded phosphate transport system grow slowly in media containing 2 mm Pi, and form nodules which fail to fix nitrogen (Fix−). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mm Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.

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