Genetic and Metabolic Engineering of Rhizobium Etli to Modify (Enhance) Nitrogen Fixation in the Symbiosis with Phaseolus Vulgaris

1999 ◽  
pp. 9-11
Author(s):  
J. Mora
Keyword(s):  
Nitrogen Fixation ◽  
Metabolic Engineering ◽  
Phaseolus Vulgaris ◽  
Rhizobium Etli

scholarly journals Effective Symbiosis between Rhizobium etli and Phaseolus vulgaris Requires the Alarmone ppGpp

2005 ◽  
Vol 187 (15) ◽  
pp. 5460-5469 ◽  
Author(s):  
Martine Moris ◽  
Kristien Braeken ◽  
Eric Schoeters ◽  
Christel Verreth ◽  
Serge Beullens ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Phaseolus Vulgaris ◽  
Regulation Of Gene Expression ◽  
Stringent Response ◽  
Rhizobium Etli ◽  
Symbiotic Interaction ◽  
Nitrogen Fixation Activity ◽  
Genome Wide ◽  
A Genome ◽  
Chain Acyl

ABSTRACT The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the σN-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific σN copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

scholarly journals Characterization of the NifA-RpoN Regulon in Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris

2010 ◽  
Vol 76 (13) ◽  
pp. 4510-4520 ◽  
Author(s):  
Emmanuel Salazar ◽  
J. Javier Díaz-Mejía ◽  
Gabriel Moreno-Hagelsieb ◽  
Gabriel Martínez-Batallar ◽  
Yolanda Mora ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Phaseolus Vulgaris ◽  
Genome Sequence ◽  
Reverse Transcription ◽  
Pcr Analysis ◽  
Rhizobium Etli ◽  
Rt Pcr ◽  
Bean Plants ◽  
Microaerobic Conditions

ABSTRACT The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alphaproteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed an R. etli CFN42 oligonucleotide (70-mer) microarray and utilized this tool, reverse transcription (RT)-PCR analysis (transcriptomics), proteomics, and bioinformatics to decipher the NifA-RpoN regulon under microaerobic conditions (free life) and in symbiosis with bean plants. The R. etli NifA-RpoN regulon was determined to contain 78 genes, including the genes involved in nitrogen fixation, and the analyses revealed 42 new NifA-RpoN-dependent genes. More importantly, this study demonstrated that the NifA-RpoN regulon is composed of genes and proteins that have very diverse functions, that play fundamental and previously less appreciated roles in regulating the normal physiology of the cell, and that have important functions in providing adequate conditions for efficient nitrogen fixation in symbiosis. The R. etli NifA-RpoN regulon defined here has some components in common with other NifA-RpoN regulons described previously, but the vast majority of the components have been found only in the R. etli regulon, suggesting that they have a specific role in this bacterium and particular requirements during nitrogen fixation compared with other symbiotic bacterial models.

A 150-megadalton plasmid in Rhizobium etli strain TAL182 contains genes for nodulation competitiveness on Phaseolus vulgaris L.

1996 ◽  
Vol 42 (9) ◽  
pp. 903-910 ◽  
Author(s):  
Dulal Borthakur ◽  
Xuefeng Gao
Keyword(s):  
Nitrogen Fixation ◽  
Phaseolus Vulgaris ◽  
Genomic Dna ◽  
Functional Complementation ◽  
Rhizobium Etli ◽  
Phaseolus Vulgaris L ◽  
Rhizobium Tropici ◽  
Hindiii Fragment ◽  
Nodulation Competitiveness ◽  
Insertion Mutants

Rhizobium etli TAL182, a competitive strain for the nodulation of Phaseolus beans, occupied more than 99% of the nodules when co-inoculated in various proportions with Rhizobium TAL1145 or Rhizobium tropici CIAT899. Two overlapping cosmid clones, pUHR68 and pUHR69, containing genes for nodulation competitiveness from TAL182, were isolated by functional complementation of strain TAL1145. Using one of these cosmid clones, we constructed two Tn5-insertion mutants of TAL182 defective in nodulation competitiveness. The Tn5 insertions in both mutants were localized in identical positions within a 4.6-kb HindIII fragment. One mutant, RUH120, was complemented for nodulation competitiveness by this HindIII fragment. The cloned DNA in pUHR68 is a part of a plasmid, 150 MDa in size, in TAL182 and does not show homology with TAL1145 genomic DNA. The 4.6-kb HindIII fragment contains a gene(s) required for nodulation competitiveness on beans, which is present only in some R. etli strains and absent in other Rhizobium spp.Key words: nitrogen fixation, competition, nodule, common bean.

scholarly journals Engineering the nifH Promoter Region and Abolishing Poly-β-Hydroxybutyrate Accumulation in Rhizobium etli Enhance Nitrogen Fixation in Symbiosis with Phaseolus vulgaris

2004 ◽  
Vol 70 (6) ◽  
pp. 3272-3281 ◽  
Author(s):  
Humberto Peralta ◽  
Yolanda Mora ◽  
Emmanuel Salazar ◽  
Sergio Encarnaci�n ◽  
Rafael Palacios ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Nitrogen Content ◽  
Seed Yield ◽  
Nitrogenase Activity ◽  
Genetic Material ◽  
Rhizobium Etli ◽  
Plant Weight ◽  
The Common

ABSTRACT Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-β-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.

Nodulation ofPhaseolus vulgarisbyRhizobium etliis enhanced by the presence ofBacillus

1997 ◽  
Vol 43 (1) ◽  
pp. 1-8 ◽  
Author(s):  
M. Srinivasan ◽  
F. B. Holl ◽  
D. J. Petersen
Keyword(s):  
Phaseolus Vulgaris ◽  
Lateral Root ◽  
Root Formation ◽  
Bacterial Species ◽  
Plant Roots ◽  
Rhizobium Etli ◽  
Phaseolus Acutifolius ◽  
Plant Interaction ◽  
Split Root ◽  
Bacillus Spp

The ability of Bacillus spp. to alter the nodulation of Phaseolus vulgaris by Rhizobium etli was assessed. The simultaneous presence of both Rhizobium etli TAL 182 and Bacillus megaterium S49 on plant roots during the early stages of plant growth was necessary for enhanced nodulation of Phaseolus vulgaris by the Rhizobium microsymbiont. Coinoculation with both bacterial species also facilitated heterologous nodulation of Rhizobium TAL 182 on Phaseolus acutifolius. These results are consistent with earlier reports of increased root hair proliferation and lateral root formation in response to coinoculation. Split-root experiments revealed that coinoculation partially suppressed host-controlled regulation of nodulation, implicating a plant interaction with the two bacterial species. Changes to the nodulation potential of R. etli due to coinoculation with Bacillus spp. demonstrate the potential for root-associated organisms other than rhizobia to alter the dynamics of the legume–Rhizobium symbiosis.Key words: Bacillus, nodulation enhancement, heterologous nodulation.

scholarly journals Novel Reiterated Fnr-Type Proteins Control the Production of the Symbiotic Terminal Oxidase cbb3 in Rhizobium etli CFN42

2007 ◽  
Vol 20 (10) ◽  
pp. 1241-1249 ◽  
Author(s):  
Manuel J. Granados-Baeza ◽  
Nicolás Gómez-Hernández ◽  
Yolanda Mora ◽  
María J. Delgado ◽  
David Romero ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Oxygen Affinity ◽  
Structural Similarity ◽  
Transcriptional Regulators ◽  
Terminal Oxidase ◽  
Nitrogen Fixing ◽  
Rhizobium Etli ◽  
High Oxygen Affinity ◽  
Key Genes

Symbiotic nitrogen-fixing bacteria express a terminal oxidase with a high oxygen affinity, the cbb3-type oxidase encoded by the fixNOQP operon. Previously, we have shown that, in Rhizobium etli CFN42, the repeated fixNOQP operons (fixNOQPd and fixNOQPf) have a differential role in nitrogen fixation. Only the fixNOQPd operon is required for the establishment of an effective symbiosis; microaerobic induction of this operon is under the control of at least three transcriptional regulators, FixKf, FnrNd, and FnrNchr, belonging to the Crp/Fnr family. In this work, we describe two novel Crp/Fnr-type transcriptional regulators (StoRd and StoRf, symbiotic terminal oxidase regulators) that play differential roles in the control of key genes for nitrogen fixation. Mutations either in stoRd or stoRf enhance the microaerobic expression of both fixNOQP reiterations, increasing also the synthesis of the cbb3-type oxidase in nodules. Despite their structural similarity, a differential role of these genes was also revealed, since a mutation in stoRd but not in stoRf enhanced both the expression of fixKf and the nitrogen-fixing capacity of R. etli CFN42.

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