David J. Sherratt

Dissection of the transposition process: A transposon-encoded site-specific recombination system

SummaryDeletions of transposons Tn1 and Tn3 that extend into a region of the transposon that specifies a 19,000 molecular weight protein, are unable to resolve presumptive transposition intermediates in recA strains of Escherichia coli. For example, when transposition of such mutant transposons occurs from replicon A to replicon B, cointegrate molecules containing A and B separated by directly repeated copies of the transposons are efficiently produced. Such cointegrates are stable in a recA strain, but are resolved within a recA+ host into replicons A and B each containing a copy of the transposon. One mutant gives cointegrates that can be complemented to resolve when a wild type Tn3 is present in the same recA cell, whereas another gives cointegrates that cannot be resolved by complementation in trans. We suggest that the first such mutant still carries the sequences necessary for the recombination event whereas the latter has lost them.The presence of a Tn1/3 specified site-specific recombination system was confirmed by showing that naturally-occurring multimers of a Tn3 derivative of plasmid pMB8 can be efficiently resolved to monomers in a recA- strain, whereas dimers of pMB9 (a Tcr derivative of pMB8) and two deleted Tn3 derivatives of pMB8 that are defective in the production of the 19,000 molecular weight protein, were both stably maintained as dimers in a recA- strain. Analysis of the ability of multimeric forms of other pMB8::Tn3 deletion derivatives to be stably propagated in a recA- strain, has allowed the localization of the Tn3 sequences necessary for the recombination event.

The transposon Tn1 as a probe for studying ColE1 structure and function.

SummaryInsertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be β-lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.

A functional map of plasmid ColE1

SummaryExamination of the properties of ColE1 derivatives containing either deletions or insertions of transposable genetic elements, has enabled a functional map of plasmid ColE1 to be constructed.

Incompatibility and transforming efficiency of ColE1 and related plasmids.

SummaryReplicons derived from the ColE1 plasmid are incompatible with one another, but are compatible with their naturally occurring relatives ColK and CloDF13. The incompatibility results in loss, by segregation, of one or the other ColE1 plasmid. In most cases, the smaller derivatives tend to displace the larger ones, and the rate of displacement depends on the difference in size. One mini-plasmid retains only 19% of the sequences of ColE1, yet it exrrts strong incompatibility: other ColE1 plasmids are rapidly lost when it is introduced into the host. The region essential for ColE1 incompatibility is deduced to lie within 700 base pairs of the origin of replication.The transforming efficiency of any ColE1 plasmid is markedly lowered when another incompatible replicon is resident in the competent cells, even when the transforming plasmid is much smaller than the resident.A model of incompatibility is proposed to account for these effects.

Synthesis of E colicins in Escherichia coli.

SummaryMitomycin C treatment of Escherichia coli cells containing one of the ColE plasmids results in specific inhibition of chromosomal protein synthesis and a high rate of protein synthesis from about 35% of the plasmid genome, whereas similar treatment of plasmid-free cells has no measureable effect on protein synthesis. In the case of ColE2-and ColE3-containing cells, the antibiotic colicin protein (molecular weight about 78000) and two others (molecular weight about 11000 and 6000) are coordinately synthesized in the approximate molar ratio 1:4:1, while in ColE1-containing cells only the colicin protein is synthesized in large amounts. Partially purified colicin E2 isolated from the outer cell surface is associated with the two small proteins in the approximate molar ratio 1:1:1, indicating that not are they only synthesized coordinately but are released as a ternary complex.

Complementation of transfer deficient ColE1 mutants

SummaryThe transfer defect of some ColE1 mutants is complemented by ColE1 or ColK, but not by ColE2. This implies that at least one ColE1-specified protein or RNA is normally needed for ColE1 conjugal transfer. The gene(s) postulated for this function lies within a region whose length is at most 50% of the genome.

ColE plasmid replication in DNA polymerase I-deficient strains ofEscherichia coli

SummaryReplication of the non-conjugative plasmids ColE1, ColE2 and ColE3 has been examined in a number of DNA polymerase I-deficient strains, two of which contain the amber mutationpolA1 along with either of two temperature-sensitivesupF amber suppressors. These latter two strains produce reduced amounts of DNA polymerase I polymerizing activity of similar, if not identical properties to that produced bypolA+ strains. Our results indicate that the ColE plasmids require different amounts of DNA polymerase I for stable plasmid maintenance. Moreover whereas all three plasmids are maintained in a strain defective in the 5′→3′ exonuclease activity of DNA polymerase I, ColE2 and ColE3 are not stably maintained between 30° and 43° in a number of DNA polymerase I-deficient strains that are temperature-sensitive for ColE1 replication.

Segregation kinetics of colicinogenic factor col E1 from a bacterial population temperature sensitive for DNA polymerase I.

SummaryWe have studied the segregation kinetics of the bacterial plasmid ColE1 from a population of cells temperature sensitive for DNA polymerase I. The results indicate that there are about twelve plasmid copies per cell of the strain used and that segregation is probably random.

Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

SummaryAfter transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.