Cloning and identification of conjugative transfer origins in the Rhizobium meliloti genome
| Autor: | José Olivares, José A. Herrera-Cervera, J. M. Sanjuan-Pinilla, Juan Sanjuán |
|---|---|
| Předmět: |
DNA
Bacterial Microbiology Genome Nucleic acid thermodynamics Plasmid Plant Microbiology Escherichia coli Genomic library Replicon Cloning Molecular Molecular Biology Cloning Genetics Sinorhizobium meliloti biology Base Sequence food and beverages Nucleic Acid Hybridization biochemical phenomena metabolism and nutrition biology.organism_classification Cosmids Conjugation Genetic Cosmid bacteria Genome Bacterial Plasmids |
| Zdroj: | Scopus-Elsevier |
| Popis: | A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer ( oriT , mob ). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriT s. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10 −2 to 10 −5 per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriT s. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10 −1 , demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae . Our results show that the R. meliloti genome contains multiple oriT s that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|