Adriana Bailone

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. In 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. The 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. A 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. In 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. From 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.

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. Using 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. The 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. The 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. The 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.

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.

Induction of recA+-protein synthesis in Escherichia coli

SummaryEscherichia coli was infected with λprecA+to determine the genetic and physiological factors controlling recA+gene expression. When λprecA+replication was prevented by superinfection immunity, recA+protein synthesis was induced by UV radiation. The recA+gene is negatively controlled by the lexA+gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA+protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA+protein synthesis. Conversely positive control of recA+gene expression requires recA+protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA+protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA+protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA+protein. λ lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA+protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA+and recA+proteins normally combine to repress the recA+gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA+protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as λ repressor.