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Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

ABSTRACT: Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.

SUBMITTER: Hernandez-Tamayo R

PROVIDER: S-EPMC3807439 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

Hernández-Tamayo Rogelio R Sohlenkamp Christian C Puente José Luis JL Brom Susana S Romero David D

Journal of bacteriology 20130809 20

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corr ...[more]

PMID: 23935046

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Project description:Conjugative transfer is one of the mechanisms allowing diversification and evolution of bacteria. Rhizobium etli CFN42 is a bacterial strain whose habitat is the rhizosphere and is able to form nodules as a result of the nitrogen-fixing symbiotic relationship it may establish with the roots of Phaseolus vulgaris. R. etli CFN42 contains one chromosome and six large plasmids (pRet42a - pRet42f). Most of the genetic information involved in the establishment of the symbiosis is localized on plasmid pRet42d, named as the symbiotic plasmid (pSym). This plasmid is able to perform conjugation, using pSym encoded transfer genes controlled by the RctA/RctB system. Another plasmid of 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 have been shown to contain endophytic bacteria from different genera and species. In this work, we have explored the conjugative transfer of the pSym (pRet42d) from R. etli CFN42 to endophytic bacteria as putative recipients, using as donor a CFN42 derivative labeled with GFP in the pRet42d and RFP in the chromosome. We were able 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. Ultrastructure microscopy analysis showed some differences in the structure of the nodules of one of the transconjugants. A replicon of the size of pRet42d (371 Kb) could not be visualized in the transconjugants, suggesting that the pSym or a segment of the plasmid is integrated in the chromosome of the recipients. These findings strengthen the proposal that nodules constitute a propitious environment for exchange of genetic information among bacteria, in addition to their function as structures where nitrogen fixation and assimilation takes place.

Project description: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.

Dataset Information

Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

Publications

Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

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