Development of molecular tools to monitor conjugative transfer in rhizobia

Tejerizo, Gonzalo Arturo Torres; Bañuelos-Vazquez, Luis Alfredo; Cervantes, Laura; Gaytán, Paul; Pistorio, Mariano; Romero, David; Brom, Susana

doi:10.1016/j.mimet.2015.08.005

Cited by 17 publications

(15 citation statements)

References 42 publications

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“…Strain CFNX559 is a derivative of R. etli CFN42 with a GFP marker in the conjugative plasmid pRet42a and an RFP marker in the chromosome. This allows differential detection of donors, which carry both markers and thus are yellow, and transconjugants, which only contain the GFP and present a green colour (Torres Tejerizo et al ., ). The experiments were done under hydroponic conditions, as described in Experimental Procedures.…”

Section: Resultsmentioning

confidence: 97%

Conjugative transfer betweenRhizobium etliendosymbionts inside the root nodule

Bañuelos-Vazquez

Tejerizo

Luz

et al. 2019

Environmental Microbiology

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Summary Since the discovery that biological nitrogen fixation ensues in nodules resulting from the interaction of rhizobia with legumes, nodules were thought to be exclusive for hosting nitrogen‐fixing and plant growth promoting bacteria. In this work, we uncover a novel function of nodules, as a niche permissive to acquisition of plasmids via conjugative transfer. We used Rhizobium etli CFN42, which nodulates Phaseolus vulgaris. The genome of R. etli CFN42 contains a chromosome and six plasmids. pRet42a is a conjugative plasmid regulated by Quorum‐Sensing (QS), and pRet42d is the symbiotic plasmid. Here, using confocal microscopy and flow cytometry, we show that pRet42a transfers on the root's surface, and unexpectedly, inside the nodules. Conjugation still took place inside nodules, even when it was restricted on the plant surface by placing the QS traI regulator under the promoter of the nitrogenase gene, which is only expressed inside the nodules, or by inhibiting the QS transcriptional induction of transfer genes with a traM antiactivator on an unstable vector maintained on the plant surface and lost inside the nodules. These results conclusively confirm the occurrence of conjugation in these structures, defining them as a protected environment for bacterial diversification.

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Section: Resultsmentioning

confidence: 97%

Conjugative transfer betweenRhizobium etliendosymbionts inside the root nodule

Bañuelos-Vazquez

Tejerizo

Luz

et al. 2019

Environmental Microbiology

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“…R. etli has two IntA-dependent recombination sites, attA present in plasmid pRetCFN42a and attD on plasmid pRetCFN42d [ 9 ]. Aiming to introduce a supernumerary att site on the chromosome, we modified a previous construction, that contained a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene [ 10 ]. This construction was modified by inserting an IntA att site between the lacZ promoter and the GFP gene.…”

Section: Resultsmentioning

confidence: 99%

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli

et al. 2016

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BackgroundThe bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system.ResultsTo fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %.ConclusionsIntegration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

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“…This 2,001-bp multicopy plasmid contains the kanamycin-resistance gene, the pBR322 origin of replication, the Trc promoter and the cloning sites Nde I and Xho I [Torres Tejerizo et al, 2015]. Genes cloned in pJOQ are expressed constitutively.…”

Section: Methodsmentioning

confidence: 99%

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Rodríguez-Mejía¹,

Roldán‐Salgado²,

Osuna³

et al. 2016

Microb Physiol

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Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

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Development of molecular tools to monitor conjugative transfer in rhizobia

Cited by 17 publications

References 42 publications

Conjugative transfer betweenRhizobium etliendosymbionts inside the root nodule

Conjugative transfer betweenRhizobium etliendosymbionts inside the root nodule

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

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