On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5.The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the “second-step-transfer” region of T5 DNA (involving the remainder of the genome). Superinfection with T4ν₁ phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.     

Dark recovery of UV-irradiated phage T1 I. A minor recovery effect whose exclusion permits the study of survival kinetics under presumably repairless conditions

The survival of UV-irradiated phage T1 is much lower in excision repair-deficient than in excision repair-proficient E. coli cells, due to lack of “host cell reactivation” (HCR). An additional decrease in phage survival occurs when repair-deficient (HCR⁻) host cells have been exposed to UV doses from 3000–10 000 erg mm⁻² of 254 nm UV-radiation prior to infection. The observed effect is attributed to loss of a minor phage recovery process, which requires neither the bacterial excision repair nor the bacterial REC repair system. This type of recovery is little affected by caffeine or acriflavine at concentrations that preclude HCR completely. Its full inhibition by UV-irradiation of the cells requires an approximately 8 times larger dose than complete inhibition of HCR.

In heavily preirradiated cells, the T1 burst size is extremely small and multiplicity reactivation is considerably less extensive than in unirradiated cells. Presumably the survival of singly infecting T1 in these cells reflects absence of any type of repair. The observed phage sensitivity and shape of the curve are compatible with the expectation for completely repairless conditions. The mechanism underlying the minor recovery is not known; theoretical considerations make a phage REC repair mechanism seem likely.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid II. Inhibition by T4 Ghosts and by T4 in the Absence of Protein Synthesis

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we showed previously that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with phage T4 and that at least two modes of inhibition can be differentiated on the basis of their sensitivity to chloramphenicol (CM). This report deals exclusively with the inhibition of E2 produced by T4, or T4 ghosts, in the absence of protein synthesis. The following observations are described. (i) The stage of T4 infection that inhibits E2 occurs after reversible adsorption of the phage to the bacterial surface, but probably prior to injection of T4 DNA into the cell's interior. (ii) The extent of inhibition increases as the T4 multiplicity is increased; however, the fraction of bacterial DNA that eventually is solubilized is virtually independent of the phage multiplicity. (iii) Phage ghosts (DNA-less phage particles) possess an approximately 15-fold greater inhibitory capacity toward E2 than do intact phage; however, because highly purified T4 (completely freed of ghost contamination) still inhibit E2, we discount the possibility that preparations of "intact phage" inhibit exclusively by virtue of contaminating ghosts. (iv) T4 infection does not liberate an extracellular inactivator of E2. In fact, infection with sufficiently high multiplicities of T4 produces a supernatant factor that protects E2 from nonspecific inactivation at 37 C. This protective factor does not interfere with the colicin's ability to induce DNA solubilization. (v) Inhibition of E2 occurs even when phage are added well after initiation of DNA solubilization by E2, suggesting that a late stage of E2 action is the target of inhibition by T4 infection. (vi) Increasing the CM concentration from 50 mug/ml to 200 mug/ml appears to reduce the inhibition appreciably; however, this can be attributed to an enhancement by CM of the rate of E2-induced DNA solubilization. (vii) The same degree of inhibition of E2 by T4 seen in CM is observed when CM is replaced by puromycin or rifampin. (viii) Others have shown that raising the multiplicity of E2 increases the rate of DNA solubilization. We find that the fractional inhibition (i), [i = (1 - y(i)/y(o)), where y(i) and y(o) represent the inhibited and uninhibited rates of solubilization of DNA, respectively], produced by a given T4 multiplicity is independent of the multiplicity of E2 and hence is independent of the rate of DNA solubilization induced by E2.     

On the lack of host-cell reactivation of UV-irradiated phage T5. I. Interference of T5 infection with the host-cell reactivation of phage T1

UV-irradiated phage T5, in contrast to T1, T3 and T7, fail to display hostcell reactivation (HCR) when infecting excision-repair proficient Escherichia coli cells. Possible causes of this lack of HCR (which T5 shares with the T-even phages) have been investigated by studying HCR of T1 under conditions of superinfection by T5. Repair-proficient B/r cells were infected at low multiplicity with UV-irradiated phage T1 in the presence of 1.8 mg/ml caffeine and were superinfected after 15 min with heavily UV-irradiated T5 amber mutants at high multiplicity. The caffeine, which is later diluted out, prevents any T1 repair prior to T5 superinfection, and UV (254 nm) irradiation of T5 with 144 J/m² reduces the ability of this phage to exclude T1, thus permitting a reasonable fraction of the mixedly infected complexes to produce T1 progeny.Under these conditions, T5 superinfection causes loss of HCR in about 90% of the T1-producing complexes. Superinfection with unirradiated T5 likewise inhibits HCR of T1, but superinfection with irradiated T3 (a host-cell-reactivable phage) does not. This indicates that the observed HCR inhibition of T1 results from T5 infection rather than from competition of irradiated foreign DNA for the excision-repair enzymes of the bacterial host. Employment of apropriate T5 amber mutants has shown that “first-step transfer” (FST) of T5 DNA (involving only 8% of the T5 genome) is sufficient for HCR inhibition, but that transfer of the remainder DNA in addition inhibits a previously described minor T1 recovery process. HCR inhibition of T1, and thus presumably lack of HCR in T5 itself, is ascribed to a substance which is produced either post infection by a gene located in the FST segment of the T5 genome, or which is transferred from extracellular T5 together with the FST DNA.     

Lysis of lysis-inhibited bacteriophage T4-infected cells.

T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption). Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes. Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed). This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities. Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption. For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation. The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed. The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.     

Effect of chloramphenicol and cyanide on the increase in UV resistance of intracellular bacteriophage T1.

The effects of chloramphenicol and cyanide on the increase in UV resistance of intracellular phage T1 infecting cells of E. coli B or E. coli Bs-1 were investigated. The inhibitiors were added to the cells 3 min prior to infection and to the complexes of phage-bacteria 3.5 and 6.5 min after adsorption of phage by the cells. The data obtained are not in agreement with the suggestion that increase in UV resistance of intracellular phage is mainly due to the accumulation of phage DNA inside the host cells. It is suggested that a very important role in this resistance is played by the interaction of phage DNA with the cell membranes.     

Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: Depolarization of the plasma membrane

Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m²) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical “exclusion”. This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).     

Bacteriophage-induced Inhibition of Host Functions: II. Evidence for Multiple, Sequential Bacteriophage-induced Deoxyribonucleases Responsible for Degradation of Cellular Deoxyribonucleic Acid

Degradation of bacterial deoxyribonucleic acid (DNA) after infection with T4 bacteriophage was studied in an endonuclease I-deficient host. The kinetics of degradation were similar to those seen in other hosts with a normal level of this enzyme. Irradiation of extracellular phage with ultraviolet (UV) destroyed the capacity of the infecting virus to induce extensive breakdown of host DNA, which was, however, converted to high-molecular-weight material. Addition of chloramphenicol to T4-infected cells provided data which can be interpreted to indicate the involvement of at least two endodeoxyribonucleases and one exodeoxyribonuclease having a high degree of specificity. A model is proposed showing the sequential action of two endodeoxyribonucleases followed by an exodeoxyribonuclease in the degradation of host DNA. The appearance of these hydrolytic enzymes requires protein synthesis. Infections leading to partial degradation only (UV-irradiated phages, gene 46 mutants) effectively inhibited the synthesis of bacterial messenger ribonucleic acid and of beta-galactosidase.     

Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation.

A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA. (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates. A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.     

Inhibition of Phage Growth by an Antibiotic Rugulosin Isolated from Myrothecium verucaria

It was found that (?)rugulosin, an antibiotic isolated from Myrothecium verucaria, had a potent anti-phage effect on RNA phages (MS2, GA and Qβ) and DNA phages (δA and T4). The effect was almost independent of the host bacterial strains used. By a detailed investigation using MS2, it was revealed that the antibiotic did not affect the free phage or host bacterium alone but inhibited phage multiplication, and the degree of the inhibition depended on the multiplicity of infection. The inhibition was not mainly due to a drop in the burst size but rather was due to a decrease of the phage-producing cells during the early stages of phage infection and replication.     

Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: Depolarization of the plasma membrane

Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m²) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical exclusion. This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.     

The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.     

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Antiphage properties of many kinds of chemicals such as antibiotics, surface-active agents and chelating agents were examined on Brevibacterium lactofermentum No. 2256—phage P465 system using double-layer agar method, as a part of the basic study, for preventing phage infection in the industrial fermentation.

A great majority of inhibitors which were selected were usually nonspecific and inhibited also bacterial growth. Among about 200 chemicals tested, 5 antibiotics such as chloramphenicol and tetracycline, 6 chelating agents such as phytic acid and 19 surface- active agents such as PEG monoester and POE alkyl ether showed the selective inhibitions for phage infection at the concentrations which did not affect bacterial growth, or at the subbactericidal concentrations that suppressed bacterial growth slightly.

Of the above chemicals which showed selective inhibitions for phage infection, a possible mechanism of chelating agents chiefly of phytic acid was investigated. When 0.1 to 0.2% of phytic acid was present in the medium, the effect of inhibition was most remarkable; this could be applied to the actual phage-infected l-glutamic acid fermentation. Phytic acid had no direct phagocidal action, nor did it inhibit the late step of the phage multiplication; but it prevented the adsorption of phages, which required inorganic cofactors such as Mg²⁺ or Ca²⁺ in this step, to the host bacteria. Moreover, a part of the infected bacteria was made incapable of forming plaques in the presence of phytic acid. These results suggested that the chelation between Mg²⁺ or Ca²⁺ and phytic acid would remove the metal ions essential for phage adsorption and prevent the phage adsorption and infection of phage DNA, consequently, the phage infection.

The effect of the non-ionic surface-active agents (SAA) on the infection of phage P465 of Br. lactofermentum was examined by adsorption and one-step growth experiments as a part of the basic study on the prevention of phage-infection in the industrial fermentation. Among various SAA tested, polyoxyethylene stearyl ether (POE-SE), polyethylene glycol monooleate (PEG-MO) and polyoxyethylene sorbitan monostearate (Tween 60) had remarkably demonstrated the selective inhibition of phage infection.

The effect of the above three SAA was apparently restricted to the initial adsorption step of phage infection, for the phage already adsorbed would not be affected by exposure to SAA. However, the results of one-step growth experiment indicated that Tween 60 inhibited not only the phage-adsorption, but also the maturation of phage already adsorbed in the host cells. The rate of the inhibition was found to be directly related to the concentration of agent. And, the most effective adsorption-inhibition was exhibited at the critical micelle concentration of SAA. The concentration as used in our experiments did not affect the viability of either phages or the host cells.

The results also indicated that the inhibition of phage-adsorption was due to the action of SAA on the surface of the bacterial cells rather than on the phage. This is supported by the observation that preincubation of phage with SAA did not affect either the subsequent adsorption rate or the plaque-forming ability of the phage. In contrast with above, a short-term exposure of bacterial cells to SAA caused an apparent change to the cell surface which was only partially restored by washing repeatedly. Moreover, the inhibitory effect of SAA on phage-adsorption appears quite specific in the phage-host system.     

Selection for lysis inhibition in bacteriophage

For Escherichia coli cells that have been infected by T-even bacteriophages (phages T2, T4, and T6), the adsorption of a second T-even phage results in an increase in the length of the original phage infection and an associated increase in the number of phages produced by the same infected cell. This is a phage encoded response called lysis inhibition. In this study the ecological significance of lysis inhibition is explored. In particular it is argued that lysis inhibition is an adaptive response to environments containing high concentrations of infected cells and low concentrations of uninfected cells.     

Canavanine-mediated depletion of polyamine pools in Escherichia coli: effect of head morphogenesis and DNA synthesis.

We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli. These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis. The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-conavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops. L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.     

Inhibierung der Entwicklung von Bakteriophagen in Fermentationsprozessen durch Zusatz von Kohlenwasserstoffen

The propagation of the virulent phage M 1 of the obligate methylotrophic bacteria strain GB 4 is inhibited by n-alkanes. The addition of hydrocarbons (7.5–22.5%) to a suspension of free phages or to a phage-host-mixture prevents the propagation of the phages, but does not influence the growth rate of the host bacteria. In a laboratory fermentor a simulated strong infection (multiplicity = 0.1) of the strain GB 4 with its phage M 1 could be suppressed by the application of 20% of a technical hydrocarbon mixture (Parex I). The inhibitory effect of the hydrocarbons can be traced back to inspecific hydrocarbon-protein-interactions at the surface of the phage and the host cells and therewith to an influencing of the adsorption.     

Bacteriophage SP82G Inhibition of an Intracellular Deoxyribonucleic Acid Inactivation Process in Bacillus subtilis

The stability of SP82G bacteriophage deoxyribonucleic acid (DNA) after its uptake by competent Bacillus subtilis was examined by determining the ability of superinfecting phage particles to rescue genetic markers carried by the infective DNA. These experiments show that a DNA inactivation process within the cell is inhibited after infection of the cell by intact phage particles. The inhibition is maximally expressed 6 min after phage infection and is completely prevented by the addition of chloramphenicol at the time of infection. The protective effect of this function extends even to infective DNA which was present in the cell before the addition of intact phage. Continued protein synthesis does not appear to be a requirement for the maintenance of the inhibition. In an analogous situation, if infectious centers resulting from singly infecting phage particles are exposed to chloramphenicol shortly after the time of infection, an exponential decrease in the survival of infectious centers with time held in chloramphenicol is observed. If the addition of chloramphenicol is delayed until 6 min after infection, the infectious centers are resistant to chloramphenicol. The sensitivity of infectious centers treated with chloramphenicol at early times after infection is strongly dependent upon the multiplicity of infection and is consistent with a model of multiplicity reactivation. These results indicate that injected DNA is also susceptible to the intracellular inactivation process and suggest that the inhibition of this system is necessary for the successful establishment of an infectious center.