Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs

A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented. Phages phi 29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated. For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr+ host. The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production. Even though phi 29 has the smallest target size of the three phages, phi 29 and its DNA are the most sensitive. Phages SPO2 and SPP1 code for gene products which complement the repair system of the host. The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr-host. This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur. The recombination process strongly interferes with the repair of damaged DNA.     

Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination.

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction.     

Bacteriophage infection in rod-shaped gram-positive bacteria: evidence for a preferential polar route for phage SPP1 entry in Bacillus subtilis

Entry into the host bacterial cell is one of the least understood steps in the life cycle of bacteriophages. The different envelopes of Gram-negative and Gram-positive bacteria, with a fluid outer membrane and exposing a thick peptidoglycan wall to the environment respectively, impose distinct challenges for bacteriophage binding and (re)distribution on the bacterial surface. Here, infection of the Gram-positive rod-shaped bacterium Bacillus subtilis by bacteriophage SPP1 was monitored in space and time. We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB, which is encoded by a putative type VII secretion system gene cluster. YueB was found to concentrate at the cell poles and to display a punctate peripheral distribution along the sidewalls of B. subtilis cells. The kinetics of SPP1 DNA entry and replication were visualized during infection. Most of the infecting phages DNA entered and initiated replication near the cell poles. Altogether, our results reveal that the preferentially polar topology of SPP1 receptors on the surface of the host cell determines the site of phage DNA entry and subsequent replication, which occurs in discrete foci.     

The minor capsid protein gp7 of bacteriophage SPP1 is required for efficient infection of Bacillus subtilis

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.     

The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.     

Phage SPP1 reversible adsorption to Bacillus subtilis cell wall teichoic acids accelerates virus recognition of membrane receptor YueB

Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis. Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compatible with an Arrhenius law. This allowed us to determine an activation energy of 22.6 kcal/mol for phage release. Finally, we show that SPP1 reversible interaction strongly accelerates irreversible binding to YueB. Our results support a model in which fast SPP1 adsorption to and desorption from teichoic acids allows SPP1 to scan the bacterial surface for rapid YueB recognition.     

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.     

Reactivation of Photoinactivated Single-Stranded DNA Bacteriophage φX174 by UV-Irradiated Escherichia coli Cells

Reactivation of single-stranded DNA phage, photodynamically inactivated in the presence of proflavine sulfate, by three isogenic Escherichia coli strains having different DNA repair capabilities has been studied. It was found that reactivation of photoinactivated phiX174 was possible only if the host cells were recombination proficient (recA(+)) and had been lightly irradiated with UV light prior to infection; the presence of the uvrA(+) gene was not essential. Only a small part of the proflavine-mediated photodynamic damage in phiX174 could be repaired in this fashion. Burst sizes of reactivated phages were, however, comparable to those of normal unirradiated phages.     

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.     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.     

On the repair mechanism responsible for multiplicity reactivation in bacteriophage T4

Summary Multiplicity reactivation has been studied using two T4 phages carrying a large number, twenty six, of genetic markers covering the whole viral genome. It has been found that in the progeny of irradiated phages the observed increase of recombination frequencies is not limited to the vicinity of radiation-induced lethal lesions, it can occur all along the phage genome. Most of the single bursts produced by multiplicity reactivation contain phages of entirely parental genotype. This fact may reveal the existence of repair by recombination between replication products of a single viral genome.     

Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site.

Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac). We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B. subtilis. Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA. The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA. Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation. The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.     

SPP1 DNA replicative forms: growth of phage SPP1 in Bacillus subtilis mutants temperature-sensitive in DNA synthesis.

Summary The development of bacteriophages SPP1, and 29 has been studied in several B. subtilis mutants defective in host DNA replication, under non permissive conditions.Several gene products, involved in the synthesis of host DNA, are required for 29 replication, while SPP1 seems to require obly the host DNA polymerase III. In addition both phages are unable to grow in a dna A mutant (ribonucleotide reductase). Taking advantage of the fact that SPP1 DNA is actively replicated in several dna mutants at non-permissive temperature, we have studied the structure of the replicative intermediates of this phage in the absence of interfering host DNA synthesis.Fast sedimenting forms of SPP1 DNA can be isolated from phage infected cells and evidence of covalently joined concatemers has been obtained, suggesting the presence of terminally repeated sequences.     

Rapid inactivation of bacteriophage T7 by ascorbic acid is repairable

Treatment of bacteriophage T7 with ascorbic acid resulted in the rapid accumulation of single-strand breaks in the DNA with double-strand breaks appearing only after incubation times of 20 min or longer. The single-strand breaks were responsible for a rapid inactivation of the phage as assayed by immediate plating of the phage-bacteria mixture on nutrient agar. Incubation of the phage-bacteria mixture in liquid medium prior to plating allowed a host cell reactivation process to repair the nicks and reactivate the phage. Non-reversible inactivation of the phage was a slower process which could be correlated with the appearance of double-strand breaks in the phage DNA. Host cell reactivation of the phage was also manifested in the phenomena of delayed lysis and delayed appearance of the concatemeric DNA replication intermediate.     

On the mutagenicity of nitrofurans

UV (254 nm)-irradiated Tr phages infecting excision-repair-proficient E. coli cells undergo host-cell reactivation (HCR). Typically, the resulting survival curve is of a hetero-component type, i.e. extrapolation of the shallower curve component to zero UV dose gives ordinate values p < 1. This characteristics is accentuated if HCR is inhibited by UV irradiation of host cells prior to phage infection, or by the presence of caffeine or acriflavine. With increasing strenght of the inhibitory condition, p decreases and the slope of the steeper curve component increases, but the slope of the shallower curve component changes very little. In contrast, single-component curves are observed for Tr infecting excision-repair-deficient host cells, or for Tr undergoing photoenzymatic repair in these cells either with or without inhibition by caffeine or preirradiation of host cells. This indicates that throughout the population UV lesions are photorepaired or photorepair-inhibited independently of one another.Discussion of various possible interpretations of the hetero-component curves in the case of HCR suggests the existence of two modes of excision repair, one of them leading to a high degree of interdependence between repair events within individual phage particles. This mode of repair, which determines the slope and position of the shallower curve component, requires within an infected cell the occurrence of a “critical event”, whose probability is considerably lower than i and decreases with the strength of HCR inhibition. The other mode, leading to independent repair events, is of minor importance under our usual plating conditions, but plays a predominant role in liquid-holding recovery of the phage.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

Susceptibility of Different Coliphage Genomes to Host-controlled Variation

Twenty-eight coliphages were studied for their susceptibility to four systems of host control variation in Escherichia coli. Both temperate and virulent phages were studied, including phages with ribonucleic acid, double- and single-stranded deoxyribonucleic acid (DNA) and glucosylated DNA. The systems examined were E. coli C-K, K-B, B-K, and K-K(P1). The C-K, K-B, and B-K systems affected temperate phages and nonlysogenizing mutants derived from temperate phages. In general, these systems did not restrict virulent phages. Phage 21e, a variant of phage 21, lost the ability to undergo restriction in the C-K and B-K systems, but retained susceptibility to the K-B and K-K(P1) systems. This suggests that the genetic site(s) on the phage, as well as in the host, determines susceptibility to host-controlled variation. Both temperate and dependent virulent phages were susceptible to the host control system resulting from the presence of prophage P1. The autonomous and small virulents were not susceptible. In a given system, the various susceptible phages differed widely in their efficiency of plating on the restricting host. If the few infections that occur arise in rare special cells, then different populations of special cells are available to different phage species. For most phage types, when a susceptible phage infected a nonrestricting host, the progeny showed the specificity appropriate to that host. Behavior of T3 was exceptional, however. When T3 obtained from E. coli K infected E. coli C or B, some of the progeny phages retained K host specificity, whereas others acquired the specificity of the new host.