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Transposition of Tn 1 to the Rhizobium meliloti genome
Abstract:
A derivative of the IncP1 plasmid RP4, carrying the thermoinducible prophage Mucts62, was obtained in Escherichia coli K12 J53 (RP4). It was impossible to maintain the hybrid plasmid RP4::Mucts62 in Rhizobium meliloti GR4. Thus, it was used as a vehicle for introducing the ampicillin-resistant transposon Tn1 into the R. meliloti genome. Transposition of Tn1 did not generate auxotrophic strains, suggesting that the insertion of Tn1 into the R. meliloti genome was relatively specific. Two chromosomal hot spots for Tn1 insertion were identified by cotransductional analysis, after general transduction by phage DF2. Plasmid-curing experiments, carried out by heat treatment, revealed that symbiotic plasmid(s) also contain at least one site for Tn1 insertion.
Insights
Researchers used a modified plasmid to introduce the ampicillin-resistant transposon Tn1 into Rhizobium meliloti. This revealed specific insertion sites in the genome and symbiotic plasmids, without causing auxotrophy.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- The IncP1 plasmid RP4 derivative, carrying Mucts62, was constructed in Escherichia coli.
- This hybrid plasmid, RP4::Mucts62, proved unstable in Rhizobium meliloti.
Purpose of the Study:
- To utilize the unstable hybrid plasmid as a vehicle for transposon delivery.
- To investigate the insertion specificity of the ampicillin-resistance transposon Tn1 in the R. meliloti genome.
Main Methods:
- Transposon mutagenesis using Tn1 delivered via the RP4::Mucts62 plasmid.
- Cotransductional analysis with phage DF2 to identify chromosomal insertion hot spots.
- Plasmid-curing experiments via heat treatment to assess Tn1 insertion in symbiotic plasmids.
Main Results:
- Transposition of Tn1 into R. meliloti did not result in auxotrophic mutants, indicating specific insertion.
- Two chromosomal hot spots for Tn1 insertion were identified.
- Symbiotic plasmids of R. meliloti were found to contain at least one site for Tn1 insertion.
Conclusions:
- The RP4::Mucts62 plasmid serves as an effective tool for Tn1 delivery into R. meliloti.
- Tn1 insertion into the R. meliloti genome exhibits site specificity, with identified chromosomal and symbiotic plasmid targets.