Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions

Girard, Lourdes; Valderrama, Brenda; Palacios, Rafael; Romero, David; Dávila, Guillermo

doi:10.1099/13500872-142-10-2847

Cited by 22 publications

(23 citation statements)

References 25 publications

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“…In R. etli, as in other rhizobia, nifHDK and other genes involved in nitrogen fixation, like fixABCX, are controlled by the oxygen-responsive NifA activator protein (22,55). Our transcriptome analysis results for symbiosis showed that all of these genes were downregulated in bacteroids of the nifA mutant strain.…”

Section: Resultsmentioning

confidence: 73%

“…We selected this time point for our study because (i) Puppo and colleagues previously found that determinate nodules like bean nodules are initiated by meristem cells in the outer cortex, but cell division stops 10 days after infection, when nitrogen fixation beings (47), (ii) Valderrama et al (55) found that the nitrogenase genes in R. etli are induced only 10 days after inoculation of the bacterium onto bean roots, and (iii) there are not drastic changes in the physiology of the nifA mutant at 11 dpi like those observed in Fix Ϫ nodules obtained at later times, for which early senescence and in most cases cell death have been documented (9,22). We demonstrated that there was nodule integrity and bacteroid viability by using electron microscopy and confocal microscopy with fluorescent dyes (propidium iodide) in nod-ules inoculated with the nifA mutant strain at 11 dpi (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The bacterial strains used were R. etli wild-type strain CFN42 (24) and mutant strain CFNX247 (⌬nifA::⍀Sp/Sm) (22). For growth under microaerobic conditions, cultures were incubated in 150-ml bottles that were closed with an airtight stoppers and flushed with several volumes of an oxygen-argon (1:99, vol/vol) mixture.…”

Section: Methodsmentioning

confidence: 99%

“…For growth under microaerobic conditions, cultures were incubated in 150-ml bottles that were closed with an airtight stoppers and flushed with several volumes of an oxygen-argon (1:99, vol/vol) mixture. The cultures were grown with shaking (200 rpm) for 10 h at 30°C as previously described by Girard et al (21,22).…”

Section: Methodsmentioning

confidence: 99%

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

Salazar

Díaz-Mejía

Moreno–Hagelsieb

et al. 2010

Appl Environ Microbiol

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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.

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Salazar

Díaz-Mejía

Moreno–Hagelsieb

et al. 2010

Appl Environ Microbiol

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“…An original version of this method was based on differential DNAcDNA hybridization of an ordered E. coli cosmid library (35), and it was applied to monitor alterations in the global transcription pattern in response to various external stimuli or regulatory mutations (7). Subsequently, modifications were made to this technique, including competitive DNA subtraction hybridization and DNA-RNA hybridization, and were used by several authors for the detection of differentially expressed, often symbiotic, genes in different rhizobia (6,9,14,22,45,46). The success of our approach is documented by the identification of three new B. japonicum genes, nrgA, nrgB, and nrgC, which are associated with a 54 -dependent Ϫ24/Ϫ12-type promoter that is activated by NifA.…”

Section: Discussionmentioning

confidence: 99%

Three New NifA-Regulated Genes in the Bradyrhizobium japonicum Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization

et al. 2000

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The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively. The product of nrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional ؊24/ ؊12-type promoter upstream of nrgA and nrgBC and proved the NifA-and RpoN ( 54 )-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

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Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives ofSinorhizobium meliloti strain 2011

Chen¹,

Higgins²,

Oresnik

et al. 2000

Electrophoresis

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Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.

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Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions

Cited by 22 publications

References 25 publications

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

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

Three New NifA-Regulated Genes in the Bradyrhizobium japonicum Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization

Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives ofSinorhizobium meliloti strain 2011

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