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.     

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein.

We cloned the gene (c1) which encodes the repressor of vegetative function of Pseudomonas aeruginosa bacteriophage D3. The cloned gene was shown to inhibit plating of D3 and the induction of D3 lysogens by UV irradiation. The efficiency of plating and prophage induction of the heteroimmune P. aeruginosa phage F116L were not affected by the presence of the cloned c1 gene of D3. When the D3 DNA fragment containing c1 was subcloned into pBR322 and introduced into Escherichia coli, it was shown to specifically inhibit the plating of phage lambda and the induction of the lambda prophage by mitomycin C. The plating of lambda imm434 phage was not affected. Analysis in minicells indicated that these effects correspond to the presence of a plasmid-encoded protein of 36,000 molecular weight. These data suggest the possibility that coliphage lambda and the P. aeruginosa phage D3 evolved from a common ancestor. The conservation of the functional similarities of their repressors may have occurred because of the advantage to these temperate phages of capitalizing on the potential of the evolutionarily conserved RecA protein to monitor the level of damage to the host genome.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Monovalent cations regulate DNA sequence recognition by 434 repressor

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage. Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins.     

Control of bacteriophage mu lysogenic repression

The transposable and temperate phage Mu infects Escherichia coli where it can enter the lytic life-cycle or reside as a repressed and integrated prophage. The repressor protein Rep is the key element in the lysis-lysogeny decision. We have analyzed the fate of Rep in different mutants by Western blotting under two conditions that can induce a lysogen: high temperature and stationary phase. We show that, unexpectedly, Rep accumulates under all conditions where the prophage is completely derepressed, and that this accumulation is ClpX-dependent. An analysis of the degradation kinetics shows that Rep is a target of two protease systems: inactivation of either the clpP or lon gene results in a stabilization of Rep. Such a reaction scheme explains the counterintuitive observation that derepression is correlated with high repressor concentration. We conclude that under all conditions of phage induction the repressor is sequestered in a non-active form. A quantitative simulation accounts for our experimental data. It provides a model that captures the essential features of Mu induction and explains some of the mechanisms by which the physiological signals affecting the lysis-lysogeny decision converge onto Rep.     

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.     

Determinants of bacteriophage 933W repressor DNA binding specificity

We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein.     

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.     

New 434-specific DNA binding protein copurified with the 434 tof protein from lambda imm434cI dv carrier cells of Escherichia coli

A tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from lambda imm434cI dv carrier cells. The apparent molecular weight of the new 434-specific DNA binding protein is 9,000 to 9,500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis. Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component. The specific binding reaction of the new protein to lambda imm434dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency. This suggests that the specific binding to lambda imm434dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself. The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation. This technique revealed that the new protein is not a fragment of the 434 tof, cII, or O protein or an NH2-terminal fragment of the cI repressor. The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.     

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.