scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

scholarly journals The Disruption of a Gene Encoding a Putative Arylesterase Impairs Pyruvate Dehydrogenase Complex Activity and Nitrogen Fixation in Sinorhizobium meliloti

2001 ◽  
Vol 14 (6) ◽  
pp. 811-815 ◽  
Author(s):  
María José Soto ◽  
Juan Sanjuan ◽  
José Olivares
Keyword(s):  
Nitrogen Fixation ◽  
Pyruvate Dehydrogenase ◽  
Sinorhizobium Meliloti ◽  
Pyruvate Dehydrogenase Complex ◽  
Chloroacetic Acid ◽  
Dicarboxylic Acids ◽  
Complex Activity ◽  
Gene Encoding ◽  
Physiological Importance

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the tri-chloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3′ end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.

scholarly journals EFFETS DES SOUCHES DE RHIZOBIUM MELILOTI ET DES COUPES SUCCESSIVES DE LA LUZERNE (MEDICAGO SATIVA) SUR LA FIXATION SYMBIOTIQUE D’AZOTE

1977 ◽  
Vol 57 (2) ◽  
pp. 433-439 ◽  
Author(s):  
L. M. BORDELEAU ◽  
H. ANTOUN ◽  
R. A. LACHANCE
Keyword(s):  
Nitrogen Fixation ◽  
Medicago Sativa ◽  
Statistical Study ◽  
Symbiotic Nitrogen Fixation ◽  
Rhizobium Meliloti ◽  
Dry Weight ◽  
Indirect Measurement ◽  
Controlled Environment ◽  
Symbiotic Efficiency ◽  
Plant Yield

Symbiotic nitrogen fixation with 49 isolates of Rhizobium meliloti was studied under controlled environment with alfalfa cv. Saranac. It was shown that plant yield in dry weight can be used as an indirect measurement of nitrogen fixation, and as a criterion for selecting efficient strains of R. meliloti. Statistical study on yields of three cuttings has established that the second cutting gives the most necessary information to correctly evaluate the symbiotic efficiency of the isolates. Six very efficient strains were selected.

scholarly journals Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

2015 ◽  
Vol 28 (12) ◽  
pp. 1353-1363 ◽  
Author(s):  
Pauline Blanquet ◽  
Liliana Silva ◽  
Olivier Catrice ◽  
Claude Bruand ◽  
Helena Carvalho ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrogen Fixation ◽  
Medicago Truncatula ◽  
Sinorhizobium Meliloti ◽  
Molecular Mechanisms ◽  
Root Nodules ◽  
Plant Protein ◽  
Post Translational Modification ◽  
Model Legume ◽  
Bacterial Mutants

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

scholarly journals HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative Symbiont Sinorhizobium meliloti

2008 ◽  
Vol 191 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Catalina Arango Pinedo ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Phosphotransferase System ◽  
Free Living ◽  
Gene Encoding ◽  
Common Component ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Living Organisms

ABSTRACT The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

scholarly journals Influence of the Poly-3-Hydroxybutyrate (PHB) Granule-Associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in Sinorhizobium meliloti Rm1021

2007 ◽  
Vol 189 (24) ◽  
pp. 9050-9056 ◽  
Author(s):  
Chunxia Wang ◽  
Xiaoyan Sheng ◽  
Raymie C. Equi ◽  
Maria A. Trainer ◽  
Trevor C. Charles ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Granule Formation ◽  
Maldi Tof ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Associated Proteins

ABSTRACT Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.

scholarly journals Cloning and Characterization of a Rhizobium leguminosarum Gene Encoding a Bacteriocin with Similarities to RTX Toxins

1999 ◽  
Vol 65 (7) ◽  
pp. 2833-2840 ◽  
Author(s):  
Ivan J. Oresnik ◽  
Sunny Twelker ◽  
Michael F. Hynes
Keyword(s):  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Bacteriocin Activity ◽  
Gene Encoding ◽  
Reading Frame ◽  
Rtx Toxins ◽  
Culture Supernatants

ABSTRACT A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in bothR. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.

scholarly journals The reaction of self-fertile alfalfa lines to inoculation with nodule bacteria

2020 ◽  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Growing Season ◽  
Dry Mass ◽  
Nodule Bacteria ◽  
Forage Crop ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Different Strains

Under the conditions of a model pot experiments, the reaction of the self-fertile lines of alfalfa Kishvardy 46, Kishvardy 27, Vertus and Ziguen to inoculation with nodule bacteria Sinorhizobium meliloti AC48 and AC88 was studied. As a result of studies, it was found that the intensity of assimilation of N2 by symbiotic systems created with the participation of various genotypes of alfalfa and active strains of S. meliloti is one of the main factors that affects the vegetative mass yield of this important forage crop. Self-fertile lines of Medicago sativa L. plants, inoculated with different strains of rhizobia were characterized by higher rates of the mass formed on the root nodules, compared to the control plants of the alfalfa variety Yaroslavna. The traditional dynamics of nitrogen-fixation activity of root nodules was maintained in all the symbiotic systems studied by us, with low values in the stems formation stage and intensive growth in the budding and flowering stages. The highest level of nitrogen fixation and vegetative growth of plants (values of plants green and dry mass, roots and root nodules mass) was established by inoculation of alfalfa line Kishvardy 46 with strain S. meliloti AC48. During the growing season the indices of the mass of nodules formed on the roots of these plants were higher by 1.8–2.3 times, the green mass by 1.2–1.6 times and the height of the plants 1.2–1.4 times as compared to the control. In the flowering stages the nitrogen-fixation activity of the symbiotic complex of plants of the Kishvardy line 27 and nodule bacteria S. meliloti AC48 exceeded the values in the symbiotic systems formed with the participation of the same strain and plants of the Ziguen and Vertus lines by 13.0 and 39.4 %. The lowest values of nitrogen fixation activity were observed by inoculation of plants of the Vertus and Ziguen lines with active strains S. meliloti AC48 and AC88 compared to the symbioses formed by the plants of the Kishvardy lines 27 and 46, as well as of the control-variety Yaroslavna with the noted strains. A stimulating effect of inoculation of alfalfa seeds of different genotypes on the growth and development of plants was noted, as evidenced by the positive dynamics of the increase in above-ground mass, the accumulation of dry matter and higher than the control values (indicators) of plant height during the growing season.

Characterisation of dry and mucoid colonies isolated from Australian rhizobial inoculant strains for Medicago species

2005 ◽  
Vol 45 (3) ◽  
pp. 151 ◽  
Author(s):  
A. McInnes ◽  
P. Holford ◽  
J. E. Thies
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Dry Weight ◽  
Sequence Element ◽  
Shoot Dry Weight ◽  
Single Colony ◽  
Agar Media ◽  
Fixation Capacity ◽  
Medicago Species ◽  
K Antigen

The presence of dry and mucoid colonies in cultures of rhizobial strains used in the production of commercial Australian inoculants is of concern for quality assurance because of the possibility of altered capacity for nodulation and nitrogen fixation by the different colony types. In this study, single colony isolates obtained from dry and mucoid colonies present in commercial cultures of Sinorhizobium meliloti were investigated to identify stability in culture, genetic identity and changes in exopolysaccharide (EPS) production, nodulation and nitrogen fixation. The 2 strains studied were WSM688 and WSM826 (Australian inoculant strains for annual and perennial medics, respectively), both of which produced only mucoid colonies on agar media when originally isolated from nodules. Dry and mucoid single colony isolates from the ‘mother cultures’ of the 2 strains exhibited stable colony phenotypes during successive subculturing in our laboratory and were shown to be most closely related to S. meliloti using 16S rRNA partial sequencing. All isolates produced at least 1 of 3 exopolysaccharides (succinoglycan, EPS II and K antigen) that are required for successful nodulation of Medicago species by S. meliloti strains, as indicated by nodulation of host legumes. Strain WSM826 isolates probably produce succinoglycan, as shown by similarity to the succinoglycan-producing strain Rm1021 in a calcofluor binding assay. In contrast to published work, there was no evidence that loss of mucoidy in dry colony isolates of either strain was associated with the presence of an insertion sequence element in the expR gene that inhibits EPS II production. For strain WSM688, dry and mucoid isolates were identical by PCR fingerprinting and showed a similar capacity to nodulate and fix nitrogen with the target host legume M. truncatula in glasshouse tests. In contrast, strain WSM826 mucoid isolates produced PCR fingerprints that were different from each other and from the WSM826 dry colony isolates. Dry and mucoid colonies may have arisen from substantial genetic change or through contamination of cultures by other S. meliloti strains. One WSM826 mucoid isolate (826-3) produced significantly lower shoot dry weight when inoculated onto both the target host M. sativa and non-target host M. truncatula, even though the capacity to nodulate both hosts was retained. This suggests that this isolate was affected in its nitrogen fixation capacity. Further research is required to identify the origin and extent of colony variation in commercial S. meliloti cultures.

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.

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