Isolation of plasmids carrying the arginine repressor gene argR of Escherichia coli K12

Summary Twenty-two sexual crosses between strains of Phycomyces blakesleeanus carrying mutations affecting phototropism (madA, madD, madE), synthesis of carotenoids (carA), auxotrophy (leu-51, nicA, pur-51), and resistance to 5-fluorouracil (fur) were studied; mating type was also included as a marker. Recombination frequencies were obtained among the ten genes involved. Linkage was found between mating type and madE; leu-51 and madA; furA401, furB402 and madD. All other gene combinations tested are unliked.     

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.     

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.     

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.     

Mutagenic DNA repair in Escherichia coli

Summary The photoreversibility of UV-induced mutations to Trp⁺ in strain Escherichia coli WP2 uvrA trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair.The results are discussed in the light of current models of UV mutagenesis.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

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.     

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.     

Curing of Escherichia coli K12 plasmids by coumermycin

Summary Low concentrations of the antibiotic coumermycin A₁, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.     

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12

Summary Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv ⁺ product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).     

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

Summary Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (oriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh ^– dnaA: Tn10 and rnh ^– oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm^–) of the rnh ^– dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA ⁺only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pasn20) in the absence of required DnaA⁺ protein nor inhibited dnaA ⁺–dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.     

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.     

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     

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.