Requirement of a Plasmid-Encoded Catalase for Survival of \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42 in a Polyphenol-Rich Environment

Nitrogen-fixing bacteria collectively called rhizobia are adapted to live in polyphenol-rich environments. The mechanisms that allow these bacteria to overcome toxic concentrations of plant polyphenols have not been clearly elucidated. We used a crude extract of polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment and analyze the response of the bean-nodulating strain Rhizobium etli CFN42. Our results showed that the viability of the wild type as well as that of derivative strains cured of plasmids p42a, p42b, p42c, and p42d or lacking 200 kb of plasmid p42e was not affected in this environment. In contrast, survival of the mutant lacking plasmid p42f was severely diminished. Complementation analysis revealed that the katG gene located on this plasmid, encoding the only catalase present in this bacterium, restored full resistance to testa polyphenols. Our results indicate that oxidation of polyphenols due to interaction with bacterial cells results in the production of a high quantity of H2O2, whose removal by the katG-encoded catalase plays a key role for cell survival in a polyphenol-rich environment

Genes encoding conserved hypothetical proteins localized in the conjugative transfer region of plasmid pRet42a from Rhizobium etli CFN42 participate in modulating transfer and affect conjugation from different donors

Among sequenced genomes, it is common to find a high proportion of genes encoding proteins that cannot be assigned a known function. In bacterial genomes, genes related to a similar function are often located in contiguous regions. The presence of genes encoding conserved hypothetical proteins (chp) in such a region may suggest that they are related to that particular function. Plasmid pRet42a from Rhizobium etli CFN42 is a conjugative plasmid containing a segment of approximately 30 Kb encoding genes involved in conjugative transfer. In addition to genes responsible for Dtr (DNA transfer and replication), Mpf (Mating pair formation) and regulation, it has two chp-encoding genes (RHE_PA00163 and RHE_PA00164) and a transcriptional regulator (RHE_PA00165). RHE_PA00163 encodes an uncharacterized protein conserved in bacteria that presents a COG4634 conserved domain, and RHE_PA00164 encodes an uncharacterized conserved protein with a DUF433 domain of unknown function. RHE_PA00165 presents a HTH_XRE domain, characteristic of DNA-binding proteins belonging to the xenobiotic response element family of transcriptional regulators. Interestingly, genes similar to these are also present in transfer regions of plasmids from other bacteria. To determine if these genes participate in conjugative transfer, we mutagenized them and analyzed their conjugative phenotype. A mutant in RHE_PA00163 showed a slight (10 times) but reproducible increase in transfer frequency from Rhizobium donors, while mutants in RHE_PA00164 and RHE_PA00165 lost their ability to transfer the plasmid from some Agrobacterium donors. Our results indicate that the chp-encoding genes located among conjugation genes are indeed related to this function. However, the participation of RHE_PA00164 and RHE_PA00165 is only revealed under very specific circumstances, and is not perceived when the plasmid is transferred from the original host. RHE_PA00163 seems to be a fine-tuning modulator for conjugative transfer.Fil: López Fuentes, Eunice. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Universidad Nacional Autónoma de México; México. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cervantes, Laura. Universidad Nacional Autónoma de México; MéxicoFil: Brom, Susana. Universidad Nacional Autónoma de México; Méxic

Genes encoding conserved hypothetical proteins localized in the conjugative transfer region of plasmid pRet42a from Rhizobium etli CFN42 participate in modulating transfer and affect conjugation from different donors

Among sequenced genomes, it is common to find a high proportion of genes encoding proteins that cannot be assigned a known function. In bacterial genomes, genes related to a similar function are often located in contiguous regions. The presence of genes encoding conserved hypothetical proteins (chp) in such a region may suggest that they are related to that particular function. Plasmid pRet42a from Rhizobium etli CFN42 is a conjugative plasmid containing a segment of approximately 30 Kb encoding genes involved in conjugative transfer. In addition to genes responsible for Dtr (DNA transfer and replication), Mpf (Mating pair formation) and regulation, it has two chp-encoding genes (RHE_PA00163 and RHE_PA00164) and a transcriptional regulator (RHE_PA00165). RHE_PA00163 encodes an uncharacterized protein conserved in bacteria that presents a COG4634 conserved domain, and RHE_PA00164 encodes an uncharacterized conserved protein with a DUF433 domain of unknown function. RHE_PA00165 presents a HTH_XRE domain, characteristic of DNA-binding proteins belonging to the xenobiotic response element family of transcriptional regulators. Interestingly, genes similar to these are also present in transfer regions of plasmids from other bacteria. To determine if these genes participate in conjugative transfer, we mutagenized them and analyzed their conjugative phenotype. A mutant in RHE_PA00163 showed a slight (10 times) but reproducible increase in transfer frequency from Rhizobium donors, while mutants in RHE_PA00164 and RHE_PA00165 lost their ability to transfer the plasmid from some Agrobacterium donors. Our results indicate that the chp-encoding genes located among conjugation genes are indeed related to this function. However, the participation of RHE_PA00164 and RHE_PA00165 is only revealed under very specific circumstances, and is not perceived when the plasmid is transferred from the original host. RHE_PA00163 seems to be a fine-tuning modulator for conjugative transfer.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

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

Background: The 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. Results: To 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 %. Conclusions: Integration 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.Fil: Hernández Tamayo, Rogelio. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Universidad Nacional Autónoma de México; México. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; MéxicoFil: Romero, David. Universidad Nacional Autónoma de México; Méxic

Genes encoding conserved hypothetical proteins localized in the conjugative transfer region of plasmid pRet42a from Rhizobium etli CFN42 participate in modulating transfer and affect conjugation from different donors

Among sequenced genomes, it is common to find a high proportion of genes encoding proteins that cannot be assigned a known function. In bacterial genomes, genes related to a similar function are often located in contiguous regions. The presence of genes encoding conserved hypothetical proteins (chp) in such a region may suggest that they are related to that particular function. Plasmid pRet42a from Rhizobium etli CFN42 is a conjugative plasmid containing a segment of approximately 30 Kb encoding genes involved in conjugative transfer. In addition to genes responsible for Dtr (DNA transfer and replication), Mpf (Mating pair formation) and regulation, it has two chp-encoding genes (RHE_PA00163 and RHE_PA00164) and a transcriptional regulator (RHE_PA00165). RHE_PA00163 encodes an uncharacterized protein conserved in bacteria that presents a COG4634 conserved domain, and RHE_PA00164 encodes an uncharacterized conserved protein with a DUF433 domain of unknown function. RHE_PA00165 presents a HTH_XRE domain, characteristic of DNA-binding proteins belonging to the xenobiotic response element family of transcriptional regulators. Interestingly, genes similar to these are also present in transfer regions of plasmids from other bacteria. To determine if these genes participate in conjugative transfer, we mutagenized them and analyzed their conjugative phenotype. A mutant in RHE_PA00163 showed a slight (10 times) but reproducible increase in transfer frequency from Rhizobium donors, while mutants in RHE_PA00164 and RHE_PA00165 lost their ability to transfer the plasmid from some Agrobacterium donors. Our results indicate that the chp-encoding genes located among conjugation genes are indeed related to this function. However, the participation of RHE_PA00164 and RHE_PA00165 is only revealed under very specific circumstances, and is not perceived when the plasmid is transferred from the original host. RHE_PA00163 seems to be a fine-tuning modulator for conjugative transfer.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in Rhizobium etli and Rhizobium leguminosarum

<p>Abstract</p> <p>Background</p> <p>A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of <it>R. etli </it>CFN42 predicts the presence of <it>panC </it>and <it>panB </it>genes clustered together on the 642 kb plasmid p42f and a second copy of <it>panB </it>on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the <it>Rhizobiales </it>with multipartite genomes, and assess the possibility that the <it>panCB </it>genes may have reached plasmids by horizontal gene transfer.</p> <p>Results</p> <p>Analysis of mutants confirmed that the <it>panC </it>and <it>panB </it>genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of <it>panCB </it>genes among members of the <it>Rhizobiales </it>showed that only <it>R. etli </it>and <it>R. leguminosarum </it>strains carry <it>panCB </it>genes in plasmids. The <it>panCB </it>phylogeny attested a common origin for chromosomal and plasmid-borne <it>panCB </it>sequences, suggesting that the <it>R. etli </it>and <it>R. leguminosarum panCB </it>genes are orthologs rather than xenologs. The <it>panCB </it>genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.</p> <p>Conclusions</p> <p>This study shows experimental evidence that core <it>panCB </it>genes located in plasmids of <it>R. etli </it>and <it>R. leguminosarum </it>are indispensable for the synthesis of pantothenate. The unusual presence of <it>panCB </it>genes in plasmids of <it>Rhizobiales </it>may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.</p

Role of plant compounds in the modulation of the conjugative transfer of pRet42a

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.Fil: Bañuelos Vazquez, Luis Alfredo. Universidad Nacional Autónoma de México; MéxicoFil: Castellani, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Luchetti, Abril. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Romero, David. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; Méxic

Transfer of the symbiotic plasmid of rhizobium etli CFN42 to endophytic bacteria inside nodules

Conjugative transfer is one of the mechanisms allowing diversification and evolution ofbacteria. Rhizobium etli CFN42 is a bacterial strain whose habitat is the rhizosphere andis able to form nodules as a result of the nitrogen-fixing symbiotic relationship it mayestablish with the roots of Phaseolus vulgaris. R. etli CFN42 contains one chromosomeand six large plasmids (pRet42a ? pRet42f). Most of the genetic information involvedin the establishment of the symbiosis is localized on plasmid pRet42d, named asthe symbiotic plasmid (pSym). This plasmid is able to perform conjugation, usingpSym encoded transfer genes controlled by the RctA/RctB system. Another plasmidof CFN42, pRet42a, has been shown to perform conjugative transfer not only in vitro,but also on the surface of roots and inside nodules, using other rhizobia as recipients.In addition to the rhizobia involved in the formation of nodules, these structures havebeen shown to contain endophytic bacteria from different genera and species. Inthis work, we have explored the conjugative transfer of the pSym (pRet42d) fromR. etli CFN42 to endophytic bacteria as putative recipients, using as donor a CFN42derivative labeled with GFP in the pRet42d and RFP in the chromosome. We wereable to isolate some transconjugants, which inherit the GFP, but not the RFP marker.Some of them were identified, analyzed and evaluated for their ability to nodulate.We found transconjugants from genera such as Stenotrophomonas, Achromobacter,and Bacillus, among others. Although all the transconjugants carried the GFP marker,and nod, fix, and nif genes from pRet42d, not all were able to nodulate. Ultrastructuremicroscopy analysis showed some differences in the structure of the nodules of one ofthe transconjugants. A replicon of the size of pRet42d (371 Kb) could not be visualizedin the transconjugants, suggesting that the pSym or a segment of the plasmid isintegrated in the chromosome of the recipients. These findings strengthen the proposalthat nodules constitute a propitious environment for exchange of genetic informationamong bacteria, in addition to their function as structures where nitrogen fixation andassimilation takes placeFil: Bañuelos Vazquez, Luis Alfredo. Universidad Nacional Autónoma de México; MéxicoFil: Cazares, Daniel. Universidad Nacional Autónoma de México; MéxicoFil: Rodríguez, Susana. Universidad Nacional Autónoma de México; MéxicoFil: Cervantes De la Luz, Laura. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez López, Rosana. Universidad Nacional Autónoma de México; MéxicoFil: Castellani, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; Méxic

Genes encoding conserved hypothetical proteins localized in the conjugative transfer region of plasmid pRet42a from Rhizobium etli CFN42 participate in modulating transfer and affect conjugation from different donors

Among sequenced genomes, it is common to find a high proportion of genes encoding proteins that cannot be assigned a known function. In bacterial genomes, genes related to a similar function are often located in contiguous regions. The presence of genes encoding conserved hypothetical proteins (chp) in such a region may suggest that they are related to that particular function. Plasmid pRet42a from Rhizobium etli CFN42 is a conjugative plasmid containing a segment of approximately 30 Kb encoding genes involved in conjugative transfer. In addition to genes responsible for Dtr (DNA transfer and replication), Mpf (Mating pair formation) and regulation, it has two chp-encoding genes (RHE_PA00163 and RHE_PA00164) and a transcriptional regulator (RHE_PA00165). RHE_PA00163 encodes an uncharacterized protein conserved in bacteria that presents a COG4634 conserved domain, and RHE_PA00164 encodes an uncharacterized conserved protein with a DUF433 domain of unknown function. RHE_PA00165 presents a HTH_XRE domain, characteristic of DNA-binding proteins belonging to the xenobiotic response element family of transcriptional regulators. Interestingly, genes similar to these are also present in transfer regions of plasmids from other bacteria. To determine if these genes participate in conjugative transfer, we mutagenized them and analyzed their conjugative phenotype. A mutant in RHE_PA00163 showed a slight (10 times) but reproducible increase in transfer frequency from Rhizobium donors, while mutants in RHE_PA00164 and RHE_PA00165 lost their ability to transfer the plasmid from some Agrobacterium donors. Our results indicate that the chp-encoding genes located among conjugation genes are indeed related to this function. However, the participation of RHE_PA00164 and RHE_PA00165 is only revealed under very specific circumstances, and is not perceived when the plasmid is transferred from the original host. RHE_PA00163 seems to be a fine-tuning modulator for conjugative transfer.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula