Plasmid replication and Hfr formation in Escherichia coli K12

Abstract

The work described here relates to the maintenance of plasmid DNA in Escherichia coli K12 by: (i) autonomous replication; and (ii) covalent integration into the host bacterial strain and subsequent replication mediated by chromosomal genes. The thesis is divided into four major sections: I. Characterisation of PB15 PB15 was formed by the integration of a mutant F’ts-gal+ plasmid into the galactose region of the chromosome of E.coli. This resulted in duplication of the gal operon, one copy being situated on either side of the sex factor genes. PB15 is an unusually stable Hfr giving rise to segregants at a frequency of <1%. To explain this low segregation frequency, Adelberg and Bergquist (1972) proposed a model which postulated that an inversion of one of the gal operons occurred after integration, trapping the episome. They demonstrated that the right-hand gal operon is not inverted, i.e. its gene order is galK, galT, galE, galO. The work reported in this thesis indicates that the left-hand gal operon is inverted. This conclusion was reached from: (i) the isolation of mutants of PB15 which had deletions extending from the right of the right-hand gal operon into the left-hand gal operon. These mutants retained functional epimerase but not functional transferase or kinase genes, indicating the left-hand gal operon is orientated -OETK - (i.e. is inverted); (ii) no gal epimerase was produced on induction of a λ lysogen of a deletion mutant such as that described above. Hence the non-informational gal strand is read on λ escape synthesis. This result also indicates that the left-hand gal operon is inverted. (iii) Gal mRNA produced on induction of a λ lysogen of this deletion mutant hybridised to the r strand of λpgal+ while gal mRNA produced on induction of a λ lysogen of a strain with a wild type-orientated gal operon hybridized to the l strand of λpgal+. II. Characterisation of seg Seg is a chromosomal gene involved in the autonomous replication of F. (i) Four temperature-sensitive seg mutations were characterised. All were shown to map at minute 89.7 on the E.co1i chromosome by P1 transduction. Seg was shown to be unrelated to other chromosomal loci located in this region which are involved in DNA replication (e.g. dnaC). The seg mutations were shown to be specific for F and λ, and no other plasmid tested was prevented from replicating in seg strains at the non-permissive temperature. (ii) Seg mutants were used to study the phenomenon of F-genote enlargement. This phenomenon had been shown to be exhibited by Hfrs arising from the integration of wild type F8-3 into a seg background and by Hfrs formed by the integration of F’ts-gal+ (F8-3) in Seg+ strains. Large chromosomal segments including F’-gal+ were transferred to recipient strains and were able to exist as independent replicons. The work reported below demonstrates that F-genote enlargement is not a particular attribute of F8-3. F-genote enlargement was shown to occur with Hfrs formed on integration of other F-primes and with the Hfrs KL983 and HfrH formed on integration of F+. III. Characterisation of Plasmid Replication Mutants Eighteen replication-defective mutants of two F’-gal+’s (F8-1 and F8-3) were characterised. These mutants fell into at least two classes on the basis of their rates of loss from cells at the non-permissive temperature. Transductional mapping with bacteriophage P1 indicated that the temperature-sensitive mutations of F8-1 are located at either 4.1Kb or 44.0Kb on the physical map of F and that the temperature-sensitive mutations of F8-3 are located at either 4.1Kb or 26.4Kb F. The simplest explanation is that both sets of mutations map at 4.1Kb F. Both sets could only map at 44.0Kb (the rep locus) if a deletion or inversion existed in F8-3. These data suggest that there are two gene clusters on F involved in autonomous replication. IV. Complementation Studies Three systems which could have allowed complementation studies to be carried out between plasmids were investigated. (i) Palchoudhury and Iyer (1971) carried out experiments which indicated that two different F-primes, F101 and F42, were able to replicate autonomously in a strain bearing the mutation dnaB43. My experiments showed that the identical dnaB43 strain does not allow complementation studies to be carried out with F42 and F101. Episomal-chromosomal interactions take place to maintain the Lac+Thr+Leu+ phenotype in Rec+ dnaB43 F-ductants containing F101 and F42 and that segregation occurs soon after the formation of heterozygotes in recAl dnaB43 strains. (ii) No complementation was observed in transient heterozygotes formed by mating F’-gal+ and F’-lac+ cells. Complementation was measured by changes in the rate of loss of a temperature-sensitive F-prime at 42°C from cells containing an introduced wild type F-prime. The results suggest that the invading F-prime does not replicate in the heterozygote and does not begin replication until segregation occurs. (iii) No complementation was observed between any of the eighteen F’ts-gal+ plasmids described above and any of the compatible plasmids ColE1, R1-16 and.Rl44-3, suggesting that these three plasmids have replication systems different from that of F.