Wilfried Wackernagel

UV-induced allevation of λ restriction in Escherichia coli K-12: Kinetics of induction and specificity of this SOS function

SummaryIn UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage λ is observed when the cells are recA+lexA+. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr+ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of λ restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting λ DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of λ infections may undergo RA.

An improved spheroplast assay for λ-DNA and the influence of the bacterial genotype on the transfection rate☆

Abstract An improved procedure for the transfection of lysozyme spheroplasts of Escherichia coli AB1157 with phage λ-DNA is described. By this method about 4 × 10 −5 infected spheroplasts and 1 × 10 −2 infective phage particles were obtained per input phage DNA molecule. With lower efficiency, transfection was also performed with DNA of phage T4. The rate of DNA uptake into the spheroplasts but not the final number of infected spheroplasts was increased if the spheroplasts did not undergo an osmotic shock from high to low sucrose concentrations during transfection. The efficiency of transfection could be further increased by using spheroplasts prepared from recombination defective recB or recC bacteria which lack an ATP-dependent nuclease probably involved in recombination ( Barbour and Clark, 1970 ). The recB- and recC -promoted enhancement of transfection efficiency could be simulated by treating rec + and recA spheroplasts (which have the ATP-dependent nuclease) with 2 × 10 −3 M KCN, an inhibitor of oxidative phosphorylation. It is therefore concluded that in rec + and recA spheroplasts about 90% of the DNA taken up is degraded by the ATP-dependent nuclease.

A mutation in Escherichia coli enhancing the UV-mutability of phage ? but not of its infectious DNA in a spheroplast assay

SummaryA mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage λ. The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage λ. However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated λ-DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.The mutation responsible for the mul phenotype has been mapped by conjugation and transduction to be located between the markers pyrE and ilv and probably marks a new gene. The mul mutation does not increase the UV-sensitivity of the cells nor does it affect their ability to perform host cell reactivation. The presence of a recA allel in a mul mutant abolishes the UV-mutability of phage λ.

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a λ phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).

Transformation and Transduction of Escherichia coli: The Nature of Recombinants Formed by Rec, RecF, and λ Red

Genetic transformation of Escherichia coli is possible when two requirements are met: (1) the recipient strain lacks exonuclease V, due to a mutation in recB or recC, but has recovered proficiency for recombination by virtue of a second suppressing mutation, sbcA or sbcB; and (2) the uptake of DNA is facilitated by treatment of the recipient cells with CaCl2 (Oishi and Cosloy, 1972; Wackernagel, 1973). However, compared with other bacteria, the frequency of transformation of E. coli is low, usually about one cell in 106. In an effort to improve this frequency we turned to a source of DNA that is both uniform and enriched for specific markers, namely the specialized transducing variants of phage λ. Our initial observations on transformation by λ gal bio DNA and our efforts to vary its genetic control suggested that certain questions might be approached more readily by studying transduction. We report here observations on transformation and transduction mediated by the RecBC and RecF pathways of E. coli (for review see Clark, 1973) and the Red system of phage λ (for review see Radding, 1973). These experiments have provided an opportunity to examine the properties of recombination mediated by the λ Red system without the possible complications introduced by maturation of phage particles.

Length determination of the terminal redundant regions in the DNA of phage T7.

SummaryThe length of the terminal redundant regions in T7 DNA has been determined by two methods. One involved the specific labeling and isolation of the redundant DNA fragment and determination of the molecular weight by polyacrylamide gel electrophoresis. A value of 150±10 nucleotide pairs was obtained. The other determination based on a correlation of the melting temperature of the redundant region to that of whole T7 DNA confirmed the result obtained by the first method.

The terminal redundant regions of bacteriophage T7 DNA

SummarySome aspects of the involvment of the terminal reduntant regions of T7 DNA on phage production have been studied by transfection experiments with T7 DNA after treatment of the molecules with λ exonuclease or λ exonuclease plus exonuclease I. It was found that terminal 5′ gaps between 0.08 and 6.4% of the total length did not decrease the infectivity of the molecules although such gaps cannot be filled directly by DNA polymerases. Rather, compared to fully native DNA the infectivity of gapped DNA increased up to 20 fold in rec+ spheroplasts and up to 4 fold in recB spheroplasts. This indicates a protective function of the single-stranded termini against the recBC enzyme in rec+ and possibly another unidentified exonuclease present also in recB. The possibility that spontaneous circularization of the gapped molecules in vivo provides protection against exonucleolytic degradation was tested by transfection with T7 DNA circularization in vitro by thermal annealing. Such molecules were separated from linear molecules by neutral sucrose gradient centrifugation. They displayed a 3 to 6 fold higher infectivity in rec+ and recB compared to linear gapped molecules, which shows that T7 phage production may effectively start from circular DNA.When the 3′ single-stranded ends from gapped molecules were degraded by treatment with exonuclease I the infectivity of the molecules was largely abolished in rec+ and recB as soon as 40 to 80 base pairs had been removed per end. It is concluded that the terminal regions of T7 DNA molecules are essential for phage production and that the redundancy comprises probably considerably less than 260 base pairs. The results are discussed with respect to the mode of T7 DNA replication.

Answers to the Problems

The phage forms a repressor. This prevents the transcription of those phage genes which are necessary to produce infectious progeny. Only found with temperent phages.

UV-sensitivity and UV-mutability of infectious ? DNA: Reactivation, protection and mutability in various assay systems

SummaryThe UV-sensitivity of phage λ and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca++-treated cells by phage DNA. It is shown that UV-irradiated λ DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr- spheroplasts and cells, resp., and since survival is also identical in Ca++-treated Hcr+ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr+ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca++-treated cells. Moreover, UV-reactivation of irradiated λ DNA, which is absent in spheroplasts, occurs efficiently in Ca++-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage λ assayed with E. coli mul-, could not be induced in λ DNA when assayed with spheroplasts or Ca++-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.

Transfer and Recombination of Genetic Material

“Recombination” means the formation of new genotypes by exchange of genetic material from two parents differing in two or more inheritable properties. In the genetics of higher organisms we differentiate between a) Recombination of “linked” genes, which lie on homologous chromosomes and can only be recombined by a crossover, and b) Recombination of “non-linked” genes, which lie on non-homologous chromosomes and therefore can be freely combined without a crossover.