Raymond Devoret

Inactivation of prophage λ repressor in Vivo

Jacob & Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product. n nIn order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants. n nThe kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case. n nA high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer. n nIn bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo. n nFrom the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.

Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

SummaryThree lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location.The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two recA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene.Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene.Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction.

Cellular levels of the prophage λ and 434 repressors

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels. n nUsing the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption. n nThe cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion. n nThe 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene. n nThe cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

SummaryIn Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD+) strains carrying mitochondrial DNA or not (rho0). This was also true for the following DNA-repair deficient (rad-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-Δ, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.

E. coli K12 inf: A mutant deficient in prophage λ induction and cell filamentation

SummaryThe bacterial mutant inf-3 (λ) is not inducible and does not form filaments following thymine starvation. Lysogenic induction is neither produced by ultraviolet light (UV) nor promoted by tif-1. This phenotype is due to a mutation infA3 located between 60 and 73 min on the E. coli K12 map.The inf mutant is resistant to X-ray and UV irradiation, in contrast to all other known non-inducible bacterial mutants. It is Rec+ and able to perform host cell reactivation as well as UV-reactivation of phage λ. After exposure to UV light, its DNA is degraded more than that of the parent and the resumption of DNA synthesis is delayed by 30 min; nevertheless, the cell survival is analogous to that of the parent. The inf mutant is also resistant to thymine starvation, for at least 3 hours.Wild type phage λ forms clear plaques on a lawn of non-lysogenic inf bacteria; a corresponding low level of lysogenization is found. The capacity of inf bacteria to reproduce phages λ, T4 or T6 is impaired.No gross defect in DNA transcription has been detected. Nevertheless, this mutant might have a slight alteration in the transcription process or in any other process involved in gene expression. This alteration might affect the regulation of DNA replication and cell division as well as prophage λ induction.

Isolation of ultravirulent mutants of phage λ

Abstract Ultravirulent mutants of phage λ able to grow in the presence of high cellular levels of repressor, have been isolated from λν2ν3. These phages can be divided into three groups of increasing virulence reflecting the number of mutational steps required for their isolation. Multiple mutations in the operator regions leading to a decrease in the affinity of the λ repressor for the operator sites seen to be responsible for the ultravirulent phenotype.

Recovery of Phage λ from Ultraviolet Damage

Recovery of phage lambda from ultraviolet damage can occur, in the dark, through three types of repair processes as defined by microbiological tests: (1) host-cell reactivation, (2) prophage reactivation, and (3) UV reactivation. This paper reviews the properties of the three repair processes, analyzes their dependence on the functioning of bacterial and phase genes, and discusses their relationship. Progress in the understanding of the molecular mechanisms underlying the three repair processes has been relatively slow, particularly for UV reactivation. It has been shown that host-cell reactivation is due to pyrimidine dimer excision and that prophage reactivation is due to genetic recombination (prereplicative). We provide evidence showing that neither of these mechanisms accounts for UV reactivation of phage lambda. Furthermore, UV reactivation differs from the other repair processes in that it is inducible and error-prone. Whether UV-damaged bacterial DNA is subject to a similar repair process is still an open question.

Construction in Escherichia coli K12 of derivatives of sex factor F143 carrying lexB and recA mutations

SummaryVarious F′ sex factors have been derived from F143, an episome extending from lysA to pheA. F143 derivatives carrying recA and lexB alleles and also mutations in genes thyA, argA, cysC were constructed as follows. Recombination was used as a means to generate genetically labelled F-primes. Using trimethoprim as agent of counterselection of Thy+ cells in thyA−/F-thyA+bacteria, it was possible to collect, after transfer, F-primes modified by deletion of the thyA region or recombination between chromosome and episome. F-primes which had spontaneously recombined with the chromosome and integrated chromosomal markers, were also selected by transfer to proper F− recipients. P1 transduction of a dominant marker allele into a strain homodiploid for a recessive allele was used to construct F-primes carrying mutations introduced by cotransduction. These F-primes have been useful to establish the dominance and complementation pattern of recA and lexB mutations (Morand, Goze and Devoret, accompanying paper; Glickman, Guijt and Morand, accompanying paper).