Inactivation of prophage lambda 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.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Proposed alpha-helical super-secondary structure associated with protein-dna recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli.

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

A novel assay for illegitimate recombination in Escherichia coli: Stimulation of λbio transducing phage formation by ultra-violet light and its independence from RecA function

We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of λ Spi⁻ phages formed during prophage induction was measured with an E. coli P2 lysogen as the indicator bacteria. Since almost all of the λ Spi⁻ phages thus detected contain attR, they have essentially the same structures as λbio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end. The following results on the formation of specialized transducing phages have been obtained by this assay system to date. (1) Irradiation with UV light greatly enhanced the formation of λ Spi⁻ phages. (2) Treatments with other DNA-damaging agents also enhanced the formation of λ Spi⁻ phages. (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of λ Spi⁻ phage formation is not controlled by the SOS regulatory system. (4) Preliminary results suggested that DNA gyrase is involved in the formation of λ Spi⁻ phage during prophage induction. Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi⁻ assay system can provide important clues to the mechanism of illegitimate recombination.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism

Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.     

Regulation in repressor inactivation by RecA protein

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate.We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-γ-S. The active ternary complex is formed by binding of ATP-γ-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-γ-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a ∅80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the ∅80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a ∅80cI-λ chimeric repressor. These results strongly suggested that th active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.     

Repressor cleavage as a prophage induction mechanism: hypersensitivity of a mutant lambda cI protein to recA-mediated proteolysis

λcIind^s prophage is hypersensitive to derepression by ultraviolet-irradiation. We have utilized this mutant to test current models for prophage derepression. We find that cIind^s repressor is cleaved by RecA protein in vivo at lower ultraviolet doses and with more rapid kinetics than cI⁺ repressor, and that induction of the recA8 gene or other LexA-repressed genes is not required for cleavage. Our results support the concept that RecA-directed proteolysis is the primary mechanism for prophage derepression.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity

Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.     

Mutations in the right operator of bacteriophage lambda: physiological effects

The right operator in bacteriophage lambda vs326 has one-twentieth the in vitro binding affinity for repressor as λv⁺; for comparison λv3 has one-quarter the affinity of λv⁺. In vivo, both mutants constitutively express genes in the right operon. Both λv3 and λvs326 express gene O constitutively because they complement λimm434Oam^? in a λ lysogen, vs, more efficiently than v3. The v3 allele in cis (but not in trans) to vs326 gives significantly greater phage yields in a λ lysogen than λvs326 alone, cro gene function, measured by arrest of exonuclease synthesis, suggested the following series of increasing degree of conatitutivity: v3, vs326, v3 vs326. λv2 vs326 forms plaques on lysogens that carry λcI857, but λv2 v3 does not. These results indicate that vs326, like v3, is an operator constitutive mutation but stronger in its effects. These mutants exemplify a uniform correlation between relative weakness of repressor binding and degree of constitutive gene expression.     

Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies.

To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.