Bernt Eric Uhlin

Physical mapping of the srl recA region of Escherichia coli: analysis of Tn10 generated insertions and deletions.

SummaryA restriction endonuclease map for the enzymes EcoRI, BamHI, SalI, and PstI covering 23.5 kilobase pairs (kb) of the srl recA region of Escherichia coli was constructed. An insertion of the transposon Tn10 in the negative regulatory gene srlR was shown to be located 5.8 kb away from the promoter roximal end of the recA gene. The extent of several Tn10 generated deletions, originating from the srlR301::Tn10 insertion, were analyzed by physical mapping. Three mutations that had removed the Tn10 encoded tetracycline resistance gene, del(srl-recA)302, del(srl-recA)304, and del(srl-recA)303, were found to be deleted for 40%, 45% and 50% of the recA structural gene, respectively. A deletion, del(srl-recA)306, that had not affected the structure of the Tn10 in srlR301 was shown to have removed the entire recA structural gene.

New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin

Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.

Nucleotide Sequence of a recA Operator Mutation lexA/Operator-Repressor Binding/Inducible Repair

SummaryA single base pair change (AT to GC) is shown to be responsible for derepression of recA. The mutation (recAo281) alters the binding sequence for the LEXA repressor.

Copy Mutants of the Plasmid R 1 as a Tool in Studies of Control of Plasmid Replication

Replication of bacterial chromosomes and plasmids is carefully regulated; their cellular concentration is kept constant in an exponentially growing population of bacteria /4/, /13/. Replication is controlled at the level of initiation /38/. The initiation mass (cell mass per origin) of the Escherichia coli chromosome is constant at least at growth rates above one generation per hour suggesting that initiation mass is the parameter used to control replication /10/, /36/, /38/. This is a reasonable idea since it requires measurement of a concentration, a principle often used in biochemical processes. Two main models have been proposed to explain the coupling between replication and growth, the one suggesting positive /19/, the other negative control /37/, /39/. Many experiments have been performed in order to distinguish between these two models, but almost no definitive answers have so far been obtained. Control of DNA replication differs from the control of synthesis of other cellular macromolecules in that the number of molecules that are synthesized per cell (and per cell generation) is limited; in some cases this number is only one. This particularly applies to the chromosome but also to many plasmids. on the other hand, it is absolutely necessary that the process is carefully controlled, otherwise cells lacking the DNA molecule could become abundant.

Effect of Plasmids on Cell Division and on the Cell Envelope of Escherichia coli

Plasmids are replicating DNA molecules that are present in a stable number of copies per cell in an exponentially growing population of bacteria (1,2). They directly affect the phenotype of their host bacteria by carrying genes mediating antibiotic resistance, bacteriocin production, catabolic functions, etc. (1,3). However, apart from these direct effects, the presence of plasmids in a cell may also indirectly affect the phenotype of the host bacterium.

Copy Number Control Mutants of the R Plasmid R1 in Escherichia Coli

DNA replication is carefully regulated in bacteria which results in a constant average number of genomes per cell in an exponentially growing population. This applies to the bacterial chromosome as well as to plasmids carried by the cells. Despite extensive studies on DNA synthesis there is very little knowledge available so far about the mechanisms involved in the regulation of DNA replication. It is generally believed that the control is exerted at the level of initiation (Pritchard, 1974). Mutant plasmids which show an increased number of copies compared to the wild type plasmid, so called copy mutants, have been isolated from several plasmids (Nordstrom et al., 1972;Tmmis and Winkler, 1973; Kool and Nijkamp, 1974; Morris et al., 1974; Matsubara and Takeda, 1975). Such plasmid mutants may be used as a tool in studies of control of DNA replication. In this paper we will report on studies with copy number control mutants of the plasmid R1 in Escherichia coli K-12.