Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

Effect of salt shock on stability of lambdaimm434 lysogens

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's cI repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage lambda(imm434). Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When lambda(imm434) lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a recA mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.     

6 (p-Hydroxyphenylazo)-Uracil: a Reversible, Selective Inhibitor of the Replication of Deoxyribonucleic Acid of Staphylococcal Bacteriophage P11-M15

6(p-Hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of the semiconservative replication of deoxyribonucleic acid (DNA) of gram-positive bacteria, was found to inhibit the replication of DNA of bacteriophage P11-M15, a virulent derivative of the temperate Staphylococcus aureus bacteriophage P11. At appropriate concentration, HPUra inhibited DNA synthesis by P11-M15-infected S. aureus immediately and completely, regardless of the stage of the lytic cycle at which infected cells were exposed to drug. The effect of HPUra was reversible since the capacity of inhibited, infected cells to replicate phage DNA and produce mature phage could be restored by removal of HPUra from incubation media. Concentrations of HPUra which completely inhibited the replication of P11-M15 in its drug-sensitive host did not inhibit the replication of this phage or its DNA in several drug-resistant host mutants. HPUra also did not inhibit the replication of two other serologically distinct, virulent staphylococcal bacteriophages, P1 and 44AHJD, in drug-sensitive hosts.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Effect of spermidine on the development of bacteriophage SP6

Bacteriophage SP6 is a virulent phage of Salmonella typhimurium which behaves differently than other phages of the same host. The effect of spermidine on SP6 infection of S. typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infection process, the replication of the phage was inhibited but the cells did not survive. Furthermore, if spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.     

Alteration of Host Specificity to Lytic Bacteriophages in Streptococcus cremoris

A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.     

Replication of Bacteriophage SH-133 in the Facultative Autotroph Hydrogenomonas facilis II. Bacteriophage Synthesis and Hydrogenase Induction Under Step-Up and Step-Down Growth Conditions

Autotrophically grown infected cells are able to replicate phage SH-133 after being switched to a heterotrophic environment (step-up growth). The effect of step-down growth on phage replication varies with the choice of organic substrate. Phage replication and the induction of cellular hydrogenase occur under step-down growth from acetate but not peptone broth. The requirement of a continued source of energy for phage replication in either heterotrophically or autotrophically grown cells could be uniquely demonstrated in this phage-host system by the deletion of hydrogen from the growth medium.     

Effects of mutations in the immunity system of bacteriophage P1.

A mutant of bacteriophage P1 that made an altered c1 repressor is described. The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action). Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens. These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity.     

Stability of the carrier state in bacteriophage M13-infected cells.

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.     

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.     

Agrobacterium tumefaciens Conn IV. Bacteriophage PB21 and Its Inhibitory Effect on Tumor Induction

Strain B2 of Agrobacterium tumefaciens Conn produces plaques when seeded against strain B6-806 of the same organism. From such a plaque, a highly virulent bacteriophage was obtained by use of D'Herelle's technique of selecting for virulent phage. On nutrient agar, this phage, PB2₁, produced large clear plaques which did not overgrow. Plaques produced on a glutamate medium and on White's plant tissue culture medium were even larger and in White's medium had a three-dimensional appearance. PB2₁ does not appear to be an oncogenic virus. To the contrary, the addition of phage under circumstances which insure mass lysis completely inhibited tumor initiation. Fewer than 10 phage particles present at the beginning of a 21-hr induction period were able, at times, to inhibit completely tumor induction by highly virulent bacteria (strain B6). The data lend further support to the concept that anything which interferes with the metabolic activity associated with the growth of the bacteria interferes with the tumor-inducing process. Attempts to use the phage to rid crown gall tissue of bacteria were unsuccessful.     

Construction of plasmids that produce phage P22 repressor

In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level. The final result of these constructions is a plasmid that maintains a level of approx. 200 times as much repressor as is found in a lysogen. A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor. The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems.     

Damaged-site independent mutagenesis of phage lambda produced by inducible error-prone repair

The existence of damaged-site independent mutagenesis is confirmed here by scoring the appearance of clear-plaque (c-) or virulent (vir) forward mutations on intact (non-irradiated) phage lambda grown on UV-irradiated E. coli K12 hosts. The mutation frequency was measured as a function of the incubation time between the occurrence of host DNA lesions and phage infection. The time course of mutagenesis of intact phage followed the induction pattern observed upon UV-reactivation of UV-damaged phage by Defais et al. (1976). Intact phage did not mutate in UV-irradiated hosts carrying the uvm-25 mutation known to prevent the occurrence of UV-reactivation. These findings suggest that damaged-site independent mutagenesis results from inducible error-prone repair. Clear-plaque mutations arising on intact phage were mostly found in phage bursts consisting of clear and turbid plaque formers whereas UV-damaged phage gave rise to mostly clear-plaque formers. Contrarily to damaged-site dependent mutagenesis, damaged-site independent mutagenesis can arise even at late times during the phage replication cycle. Our data indicate that about half of the phage mutations that arise upon UV-reactivation are damaged-site independent mutations. Replication of intact phage DNA in a host during induction of SOS functions provides a sensitive assay for the detection of damaged-site independent mutagenesis.     

Method for isolating restriction- and modificationless mutants of Escherichia coli K-12.

A simple method is described for the selection and isolation of restriction- and modificationless mutants in Escherichia coli K-12 by using the following properties: (i) the temperature-sensitive repressor activity of phage lambdacI857; (ii) a mutant of lambda phage defective in integration and the establishment of repression (lambdab2cI); (iii) a virulent lambda phage insensitive to the repressor activity. The final yield of spontaneously arising rk-mk+ and rk-mk- mutants from stationary-phase cultures was about 5% of the surviving cells.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

Lysogenization by bacteriophage lambda: II. - Identification of genes involved in the multiplicity dependent processes

We previously showed that, under conditions of rapid exponential growth, lysogenization of E. coli cells by phage λ requires that the cell is infected by at least 2 phages able to replicate their DNA, or 3 or 4 phages unable to replicate their DNA [ref. 4]. Since genes dealing with prophage integration appear not to be involved in these multiplicity dependent processes, a determination was made as to whether more than one copy of the genes involved in repressor synthesis or its activation are needed for lysogenization. The complementation patterns which we obtained indicate multiplicity effects involving gene cII (and, perhaps, cIII) in lysogenization by both phage able or unable to replicate. In the former case, we propose that cII protein (and, perhaps, cIII) both induces repressor synthesis and inhibits phage DNA replication. In lysogenization by phage unable to replicate, the data suggest that the expression of early phage genes and repressor synthesis in the course of lysogenization are mutually exclusive processes which do not take place on the same phage chromosome.     

Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu.

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.