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Title: A genetic analysis of conjugation by the IncP-1 plasmid RP1
Author: Schmidt, Londa
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1982
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The IncP-1 plasmids are of interest because of their ability to transfer into and be stably maintained in a large variety of gram negative genera. This ability is of interest because of both the evolutionary and medical implications arising from the existence of such plasmids in nature. In this thesis I have begun a genetic analysis of the transfer function of the IncP-1 plasmid RP1 (this function has been shown to be located in 3 distinctly separate areas of the plasmid). RP1 was mutagenised to isolate transfer deficient (Tra ) mutants of the RP1 plasmid. Of the 133 Tra mutants selected 97 were resistant to the IncP-l-specific phages PRR1 Pf3 and PR4. While 6 had a PRRlr Pf3{ PR4s phenotype and 30 were sensitive to the phages. Twenty one of the mutants had amber mutations of Tra; of these, 19 were RPi-specific phage resistant and two were PRRlr Pf3r PRO. None of the RP1 amber Tra mutants were sensitive to the 3 RP1-specific phages tested. Mu phage has been shown to both integrate randomly into a genome and cause mutations. Mil phage was used to try to obtain RP1 Tra-mutations caused by insertion of Mu into a Tra region. Although none of the RP1::Mu lysogens were Tra, several were of interest. Some had Mu inserted close or adjacent to Tra region of RP1 and could be used to generate Tra- deletions upon induction of the phage. These deletion mutants would be helpful in ordering representative point mutants within the Tra regions. To begin grouping the Tra mutants into complementation groups, transient hetarozygote complementation experiments using both amber mutants in a Su+ strain as donors and P1 transducing phages of RP1 Tra- mutants were attempted. These results were unsatisfactory; probably because the RP1 transfer frequencies in liquid medium are low. Qualitative transient heterozygote complementations using a replica-plate method allowed the amber RP1 fro mutants to be grouped into 10 complementation groups. Cloning of transfer genes away from the incompatibility determinants has been used to advantage in the study of the F transfer system. Therefore, the RP1 Tral region was cloned into the plasmid vector pBR325 using an RP1::Tn7 derivative of RP1. This chimera (pED800) was used to make partial diploid strains with all of the amber Tra- mutants. All the Tra mutants which showed sensitivity to the specific phages were complemented by pED800. Two of the amber Tra- mutants which were phage resistant were also complemented by pED800. Since the majority of the Tra- mutants tested were RP1-specific phage resistant and not complemented by pED800, it was concluded that most of the tra genes are not found in the Tral region. ITra homogenote derivatives of pED800 carrying the pED516 (PP.R1t Pf3r PRO amber Tra-) and pED615 (PRRlr Pf3r PR4r amber Tra) mutations were isolated. These derivatives were used to make partial diploid strains with some of the mutants which were complemented by pED800 and the diploid strains were tested for complementation. On the basis of these complementation experiments it was established that at least 4 tra genes are found in the Tra1 region of RP1. The Tra2 and Tra3 regions were not cloned due to lack of selecting markers and time. Nalidixic acid was found to interfere with RP1 conjugation to a similar extent as it did with F conjugation. This was probably due to inhibition of DNA replication by interfering with the functions of DNA gyrase. At least 20 plasmid coded proteins were found to be synthesized by RP1 in 35S-methionine labeled minicells. Once the Tra cistrons have been identified it will be possible to determine which of these proteins are RPl Tra proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available