Requirement of DNA Gyrase for the initiation of chromosome replication in Escherichia coli K-12

Summary It has been found that strains carrying mutations in the dnaA gene are unusually sensitive to COU, NAL or NOV, which are known to inhibit DNA gyrase activities. The delay in the initiation of chromosome replication after COU treatment has been observed in cells with chromosomes synchronized by amino acid starvation or by temperature shift-up (dnaA46). The unusual sensitivity of growth to COU of the initiation mutant runs parallel to a higher sensitivity to the drug of the initiation of chromosome replication.The double mutant, dnaA46 cou-110 has been isolated and mutation cou-110 conferring resistance of growth, initiation and elongation of chromosome replication to COU was mapped in the gene coding for the subunit of DNA gyrase. The reduced frequency of appearance of the mutants resistant to COU, NAL or NOV in the initiation mutant suggests that some mutations in genes coding for DNA gyrase subunits cannot coexist with the dnaA46 mutation. The possible mechanisms of the requirement of DNA gyrase for dnaA-dependent initiation of E. coli chromosome are discussed.Abbreviations used COU coumermycin A₁ - NAL nalidixic acid - NOV novobiocin     

DNA degradation in minicells of Escherichia coli K-12

Summary The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC^- phenotypes are unaffected by min ⁺/^- genotypes in whole cells. In contrast to minicells produced by rec ⁺ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or dv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [³²P]-labelled linear DNA from bacteriophage T₇ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec ⁺ and recA^- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC^- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.Paper 1 in series, see Khachatourians et al., 1974.     

Inhibition of initiation of mini-F plasmid replication in temperature-sensitive mafA mutants of Escherichia coli K-12

When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [³H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Initiation of DNA replication in Escherichia coli

Summary When E. coli F⁺ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F⁺ dna-167 strain, but can do so in the F⁺ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Hyper-recombination in uvrD mutants of Escherichia coli K-12

Summary A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.     

Interplasmidic and intraplasmidic recombination in Escherichia coli K-12

Summary The construction of plasmids which facilitate the study of interplasmidic and intraplasmidic recombination is described. In this system, a single recombination event between two mutated Te^r genes on separate plasmids or on one plasmid leads to a change in the host phenotype from sensitivity to resistance to tetracycline.Recombination proficiencies have been determined for different E. coli K-12 strains: both interplasmidic and intraplasmidic recombination are independent of the recBC gene product. RecA mutations decrease the proficiency of plasmidic recombination 40–100 fold. Intraplasmidic and interplasmidic recombination via the recE pathway are more efficient than via the recBC pathway. Intraplasmidic recombination, but not interplasmidic recombination via the recE pathway is independent of a functional recA product.     

The process of general recombination in Escherichia coli K-12

Summary A review of the data on the genetic determination of general recombination in Escherichia coli introduces three alternative pathways of recombination, RecBC, RecF, and RecBCF. One recBC-dependent pathway is functional in recF ^– cells. An initiating endonuclease is involved, acting on the chi-sites of DNA. The second is recF-dependent, acting in the double mutant recBC sbcB. The corresponding endonuclease uses the fre-sites as a substrate. A third pathway acting in wild-type cells is mixed. Both enzymatic systems participate in the overall process. We shall call it RecBCF.Using the thermosensitive recA44 mutant it became possible to study the kinetics of integration of donor DNA into the recipient chromosome via the RecF and RecBCF pathways of recombination. The RecF pathway is characterized by delayed recombination; not less than 14 h being needed to complete the process at 35° C. By the RecBCF pathway (wild-type recipient) the reaction is fast, as described by Lloyd and Johnson (1979). The two stage nature of the RecF pathway is important. First an intermediate product is formed during a short time interval. This product is resistant to the degrading exonuclease V. Afterward the intermediate product is slowly integrated into the recipient chromosome. Autoradiography of this intermediate product, extracted from exconjugants, shows that it consists of closed DNA circles. Their length is within the limits 2–15 min on the E. coli map. Their average value is in fair agreement with genetic estimations of the integrated DNA fragments.Taking into consideration the similarity between genetic determinations of the fre-effects and the heterogeneity of the progeny, we conclude that the intermediate structures formed contribute to this heterogeneity.     

Deletion map of the Escherichia coli K-12 dnaB gene

Summary Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P ₂ and their total absence in mutant P _a. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either -naphthyl phosphate or different acetate esters as substrates.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Summary Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.     

A new radiation sensitive mutant of Escherichia coli K-12

Summary Streptomycin treatment of competent cultures of Bacillus subtilis kills the non competent cells. This method was used to look for mutagenic effects of Streptomyces coelicolor DNA. This DNA was found to induce a class of met⁺ clones harbouring aromatic adventitious mutations.     

A temperature-sensitive mutant in prolinyl-tRNA ligase of Escherichia coli K-12

Summary A mutant with a defective prolinyl-tRNA ligase has been found in a collection of spontaneous temperature-sensitie mutants. The mutated gene, which is designated proS, is closely linked to metD.     

Irreversible thermal denaturation of uridine phosphorylase from Escherichia coli K-12

Thermal denaturation of uridine phosphorylase from Escherichia coli K-12 has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were obtained at temperature scanning rates of 0.25, 0.5, and 1 K/min. These profiles were analysed using three models of irreversible denaturation which are approximations to the whole Lumry-Eyring model, namely, the one-step model of irreversible denaturation, the Lumry-Eyring model with the fast equilibrating first step, and the model involving two consecutive irreversible steps. In terms of statistics the latter model describes the kinetics of thermal denaturation of uridine phosphorylase more satisfactorily than the two other models. The values of energy activation for the first and second steps calculated for the model involving two consecutive irreversible steps are the following: E_a,1 = 609.3 ± 1.8 kJ/mol and E_a,2 = 446.8 ± 3.2 kJ/mol.