Program Responsible: Dr. David Romero.
- Recombination pathways in Rhizobium etli.
- Mechanisms of gene conversion in the Rhizobium genome.
- Generation of tools for genome engineering in Rhizobium.
- Systematic analysis of Rhizobium plasmids by programmed deletions.
- Regulation of conjugative transfer of the symbiotic plasmid of Rhizobium etli.
- Search for the presence of site-specific recombination systems, similar to the one we found on R. etli CFN42 plasmids, on other Rhizobiacea.
- Identification of plasmid-encoded genes involved in the adaptation of Rhizobium etli to the rhizosphere of Phaseolus vulgaris using genetic and genomic approaches.
- Role of the extracitoplasmic (ECF) sigma factors in Rhizobium etli.
This line is focused on the identification and characterization of recombination genes in this bacterium. We have isolated different components of the R. etli recombination machinery, like recA, addAB, ruvABC, recF recG, radA and mutS. These genes are relevant for the generation of genome rearrangements, typical feature of this organism. Moreover, all these genes are important in maintaining the genome integrity and in repairing damaged DNA.
In this line, we study the process of gene conversion occurring between repeated sequences in the Rhizobium genome. This process is particularly interesting, because it can explain the high identity in nucleotide sequence among repeated sequences, achieved through concerted evolution. Our work is devoted to understanding the mechanisms involved in its generation, in the context of the Double-Strand Break Repair Model. We are currently studying the effect of mutations in proteins involved in recombination on gene conversion, including initiation of recombination (addAB and recF), proofreading (mutS) and migration of the Holliday intermediate (ruvAB, recG and radA)
Participants: Mildred Castellanos, César Rodríguez, Jaime Martínez, David Romero
Genome Engineering is defined as the in vivo modification of the genome, using homologous or site-specific recombination. In this area, we are taking advantage of our previous experience in selection of genome rearrangements. Moreover, we are implementing several tools for the generation of programmed deletions (using the Cre-loxP system), “erasable marker” technology (also using the Cre-loxP system), efficient introduction of point mutations (using gene conversion) and efficient introduction of marked mutations (using recombineering or approaches based in the in vivo delivery of double-strand cuts).
Continuing with our interest in functions encoded in Rhizobium plasmids, we are generating a collection of programmed deletions in each of the six large plasmids of Rhizobium etli. Strains resulting from this work will be used in experiments of functional genomics.
In this line, we are studying the mechanisms involved in repression of a conjugative system present on the pSym of R. etli. This system, which employs one repressor gene (rctA) also has interactions with another gene product (RctB) whose overproduction apparently overrides the repression imposed by RctA. Characterization of the regions relevant for transcriptional repression as well as purification and analysis of RctA and RctB are the objectives of this line.
Search for the presence of site-specific recombination systems, similar to the one we found on R. etli CFN42 plasmids, on other Rhizobiacea.
Our previous work has shown the participation of a site-specific recombination in the formation or resolution of cointegrates between the symbiotic plasmid and a smaller, self-transmissible plasmid. These cointegrates are important to achieve conjugative transfer of the pSym. Our work will continue with the search of similar elements on other Rhizobiaceae. This work will contribute to a better understanding of the role of plasmids in the rhizobial life cycle, through the knowledge of plasmid encoded functions, and of the mechanisms that contribute to the distribution of symbiotic plasmids and the elements and conditions that control these processes.
Identification of plasmid-encoded genes involved in the adaptation of Rhizobium etli to the rhizosphere of Phaseolus vulgaris using genetic and genomic approaches.
We are looking for genes involved in the adaptation of Rhizobiumetli CFN42 to the rhizosphere of Phaseolus vulgaris. The genome of this bacterium consists of one circular chromosome (4.3 Mb) and six plasmids (p42a–p42f) ranging in size from 184 to 640 kb. We have isolated derivatives of the parental strain cured of each plasmid as well as cured of multiple plasmids. Co-inoculation experiments on bean plants demonstrate that derivatives cured of each plasmid are significantly less competitive for root nodulation than the wild type strain. The most drastic decrease in competitivity for nodulation has been observed in a multiple plasmid-cured derivative. These data indicate that plasmid-encoded genes play an important role in the adaptation of R. etli to the rhizosphere of bean plants.
Since the genome of this bacterium has been totally sequenced, we are using a combination of genetics and genomics approaches to identify and characterize plasmid-encoded genes involved in the adaptation of R. etli to the rhizosphere of bean plants. These experimental strategies include:
- Complementation of plasmid-cured derivatives with a genomic library of R. etli.
- Interposon inactivation of predicted genes looked up in the genome annotation derived from the R. etli genome project.
- The use of a genome-engineering tool based on the Cre/loxP integration-excision system to delete large plasmid fragments (100 kb).
Participants : Laura Cervantes, Susana Brom, Erika López López, Alejandro García
In this line we are interested in knowing the role of the ECF sigma factors, subunits of the RNA polymerase that recognize specific promotors of genes to be transcribed (sigmulon). The ECF factors are involved in envelope stress response, for example, a member of this group regulates the iron uptake, oxidative and heat shock in E. coli, alginate production and cyst formation in A. vinelandii, exotoxin production and oxidative stress in P. aeruginosa, etc. In R. etli there are about 18 ECF-factors and their role is unknown, therefore we are studying their role in the legume-bacteria interaction process , in different stresses (like oxidative, osmotic, heat shock) and in free-living conditions. In order to do that, we are determining the conditions that allow the gene expression for each ECF sigma factor as well as constructing mutants that will allow us to evaluate their biological function.