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.     

Weigle reactivation of the single-stranded DNA phage f1

Summary The survival of ultraviolet light (UV) damaged single-stranded DNA bacteriophage f1 is increased when the Escherichia coli host is irradiated with UV prior to infection. This repair, called Weigle reactivation, is multiplicity independent and is absent in recA and in lexA mutants. The function of the recA-lexA repair system needed is repair and not recombination, as demonstrated by the absence of Weigle reactivation in mutants that are recombination proficient but defective in repair of double-stranded DNA. Weigle reactivation of f1 requires high levels of the recA protein, and in addition activation of recA or another protein. This activation can be produced by UV irradiation, or by the tif-1 allele of recA together with the spr allele of lexA. Mutagenesis of f1 has the same requirements as W-reactivation, and in addition requires UV irradiation of the phage.     

SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA 2 ′ B, but not UmuD 2 ′ C (PolV) polymerase

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength >320 nm are increased when the wild-type host (Escherichia coli uvr ⁺) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as “W reactivation” and “W mutagenesis.” It is shown that 8-MOP monoadducts in λ DNA induce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA ₂ ^′ B, but not PolV (UmuD ₂ ^′ C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc ⁺ in E. coli uvr ^?.     

W-reactivation of phage lambda in X-irradiated mutants ofEscherichia coli K-12

Summary The survival of UV irradiated phage lambda was increased on X-irradiatedE. coli K-12 host cells over that on unirradiated cells. The frequency of c mutants among the surviving phages was to a similar extent increased by the X-ray exposure of the host cells as by UV light. This W-reactivation of phage lambda occurred inuvrA, polA, andrecB mutants besides the wild type at about equal X-ray doses, however, at a reduced reactivation efficiency compared with the wild type. W-reactivation was undetectable inrecA mutants. While maximal UV induced W-reactivation occurred 30 min after irradiation, the maximal X-ray induced reactivation was found immediately after irradiation. Chloramphenicol (100 µg/ml) and nitrofurantoin (50 µg/ml) inhibited W-reactivation of phage lambda if added before irradiation of the host cells, indicating the necessity of protein synthesis for W-reactivation.     

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

Summary The ability to reactivate ultraviolet (UV) damaged phage CbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage CbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2–2 h at 30°C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5–10 J/m² of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.     

Inducible, error-free DNA repair in tsl recA mutants of E. coli

Summary Host cell reactivation and UV reactivation and mutagenesis of UV-irradiated phage were measured in tsl recA ⁺ and tsl recA host mutants. Host cell reactivation was slightly more efficient in the tsl recA strain compared to the tsl ⁺ recA strain. Phage was UV-reactivated in the tsl recA strain with about one-half the efficiency of that in the wild type strain, but there was no corresponding mutagenesis of phage. UV-reactivation was also slightly lower and mutagenesis several-fold lower than normal in the tsl recA ⁺ strain. To account for these observations, we propose that there is an inducible, error-free pathway of DNA repair in E. coli that competes with error-prone repair for repair of phage lesions.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria

Summary The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex ⁺ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression.Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and uv-induced mutagenesis.

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol^?) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol^? mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol⁺ parent. The presence of FP18 or FP111 in the Pol^? mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant.The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla.The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.     

Transfection of Vibrio cholerae by bacteriophage phi 149 DNA

DNA isolated from Choleraphage ø149 of Group IV was infectious when mixed with competent $\text{V.}\text{cholerae}$ cells. The cells were competent during mid-log phase of growth. The infectivity of phage DNA was destroyed by deoxyribonuclease but not by ribonuclease or pronase. About 5 min is required for the establishment of the DNase resistant state. The dose response curve for transfection suggested that 2 to 3 molecules of DNA are required to produce one infections center. An infectivity of 5 × 10⁴ infectious center per μg of DNA was obtained.     

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.     

Reactivation of Giardia lamblia cysts after exposure to polychromatic UV light

Aims: In this study, we determined the ability of a promising alternative UV technology – a polychromatic emission from a medium‐pressure UV (MP UV) technology – to inhibit the reactivation of UV‐irradiated Giardia lamblia cysts. Methods and Results: A UV‐collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS (pH 7·2) or filtered drinking water to a low dose (1 mJ cm^?2) of MP UV irradiation. After UV irradiation, samples were exposed to two repair conditions (light or dark) and two temperature conditions (25°C or 37°C for 2–4 h). The inactivation of G. lamblia cysts by MP UV was very extensive, and c. 3 log₁₀ inactivation was achieved with a dose of 1 mJ cm^?2. Meanwhile, there was no apparent reactivation (neither in vivo nor in vitro) of UV‐irradiated G. lamblia under the conditions tested. Conclusion: The results of this study indicated that, unlike the traditional low‐pressure (LP) UV technology, an alternative UV technology (MP UV) could inhibit the reactivation of UV‐irradiated G. lamblia cysts even when the cysts were exposed to low UV doses. Significance and Impact of the Study: It appears that alternative UV technology has some advantages over the traditional LP UV technology in drinking water disinfection because of their high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV‐irradiated G. lamblia cysts.     

Inactivation of transfecting DNA by physical and chemical agents: influence of genotypes of phage lambda and host cells

Inactivation of λ₁₁c and its purified DNA by UV irradiation, γ-rays of ¹³⁷Cs (in conditions of indirect action), nitrous acid, hydroxylamine and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied. The biological activity of isolated phage DNA was measured by the calcium transfection procedure. 14 different recipient strains of Escherichia coli K12 were used, including mutants deficient in excision and recombination repair (uvrA6, uvrB5, uvrC34, polA1, recA13, recC38, recD34, recA13B21C22, recA56uvrA6, exrA and recB21C22sbcB15).Whole phage was more resistant to the action of γ-rays than was isolated DNA. On the other hand, the chemical agents HNO₂ and MNNG inactivated phage much faster than isolated DNA. Of all mutations of the host cell only polA1 considerably increased the sensitivity of phage DNA to UV irradiation, γ-rays and MNNG. The mutations uvr^? affected the inactivation kinetics under UV action. In all other cases the genotype of the host cell was indifferent for the inactivation kinetics of phage DNA, even if it belonged to recombination deficient mutant λ red3 int6 (in which only UV and γ inactivation was studied). Possible reasons for the low efficiency of the host-cell repair toward the damage caused to λ DNA by different agents are discussed.     

Host Cell Reactivation in Strains of <Emphasis Type="Italic">E. coli</Emphasis> lacking DNA Polymerase Activity <Emphasis Type="Italic">in vitro</Emphasis>

BACTERIAL cells can repair DNA which has been damaged by irradiation with ultraviolet light (UV). A repair process which does not depend on light is known as dark reactivation. Because the cells can repair damaged infecting DNA, as well as their own by the same mechanism the phenomenon has also been called host cell reactivation (HCR). HCR seems to consist of the following reaction steps¹: (1) endonucleolytic incision close to the UV photoproduct which most frequently is a pyrimidine dimer; (2) excision of the photoproduct as an oligonucleotide; (3) resynthesis of the removed nucleotide sequence using the opposite strand as a template; and (4) rejoining of the polynucleotide chains.     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

Morphological Features of a Filamentous Phage of Vibrio cholerae O139 Bengal

A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 μm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10⁸ to 10⁹ pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.     

Ultraviolet inactivation and photoreactivation of the cholera phage ‘Kappa’

Summary The lysogenic cholera phage, Kappa is some ten to twenty folds more resistant to UV (254 nm) than are most of the T. phages ofE. coli, or the cholera phage PL 163/10, or the hostV. cholerae strain H218 Sm^r, the 37% (D ₃₇) and 10% (D ₁₀) survival doses being 255.8 J/m² and 633.6 J/m² respectively. The UV-irradiated Kappa phages could be photoreactivated in the hostV. cholerae strain H218 Sm^r to a maximum extent of 40%. The removal of the number of lethal hits per phage by the survival-enhancement treatment (photoreactivation) with time followed an exponential relation, the constant probability of removal of lethal hit per unit time being 2.8 × 10^–2 min^–1. The UV-irradiated phages could also be Weigle reactivated in the host strain H218 Sm^r by a small degree, the maximum reactivation factor (ratio of survivals in UV-irradiated and non-irradiated hosts) being 1.50.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.