The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting Prophages.

Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The effects were dramatic in Shiga toxin-converting phage Φ24(B) after treatment with oxidative stress-inducing agent, hydrogen peroxide, while they were less pronounced in bacteriophage λ and in both phages irradiated with UV. The hydrogen peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells lysogenic for either λ or Φ24(B), deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R) could be observed in Φ24(B)-, but not in λ-bearing cells. We conclude that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and regulatory genes of Shiga toxin-converting bacteriophage Φ24(B).

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1.

UNLABELLED: Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so far the only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as a molecular switch for the transcriptional regulation of the early viral genes. IMPORTANCE: Functional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.

Probability landscape of heritable and robust epigenetic state of lysogeny in phage lambda.

Computational studies of biological networks can help to identify components and wirings responsible for observed phenotypes. However, studying stochastic networks controlling many biological processes is challenging. Similar to Schrödinger's equation in quantum mechanics, the chemical master equation (CME) provides a basic framework for understanding stochastic networks. However, except for simple problems, the CME cannot be solved analytically. Here we use a method called discrete chemical master equation (dCME) to compute directly the full steady-state probability landscape of the lysogeny maintenance network in phage lambda from its CME. Results show that wild-type phage lambda can maintain a constant level of repressor over a wide range of repressor degradation rate and is stable against UV irradiation, ensuring heritability of the lysogenic state. Furthermore, it can switch efficiently to the lytic state once repressor degradation increases past a high threshold by a small amount. We find that beyond bistability and nonlinear dimerization, cooperativity between repressors bound to O(R)1 and O(R)2 is required for stable and heritable epigenetic state of lysogeny that can switch efficiently. Mutants of phage lambda lack stability and do not possess a high threshold. Instead, they are leaky and respond to gradual changes in degradation rate. Our computation faithfully reproduces the hair triggers for UV-induced lysis observed in mutants and the limitation in robustness against mutations. The landscape approach computed from dCME is general and can be applied to study broad issues in systems biology.

Isolation of Phage via Induction of Lysogens.

Most bacterial cells carry prophage genomes either integrated into the host DNA or present as repressed plasmids. Methods are described for the induction of prophages using Mitomycin C, and for the isolation of prophage-cured bacterial cell lines.

Cultivation-based assessment of lysogeny among soil bacteria.

Lysogeny has long been proposed as an important long-term maintenance strategy for autochthonous soil bacteriophages (phages). Whole genome sequence data indicate that prophage-derived sequences pervade prokaryotic genomes, but the connection between inferred prophage sequence and an active temperate phage is tenuous. Thus, definitive evidence of phage production from lysogenic prokaryotes will be critical in determining the presence and extent of temperate phage diversity existing as prophage within bacterial genomes and within environmental contexts such as soils. This study optimized methods for systematic and definitive determination of lysogeny within a collection of autochthonous soil bacteria. Twenty bacterial isolates from a range of Delaware soil environments (five from each soil) were treated with the inducing agents mitomycin C (MC) or UV light. Six isolates (30%) carried inducible temperate phages as evidenced by an increase in virus direct counts. The magnitude of induction response was highly dependent upon specific induction conditions, and corresponding burst sizes ranged from 1 to 176. Treatment with MC for 30 min yielded the largest induction responses for three of the six lysogens. Morphological analysis revealed that four of the lysogens produced lambda-like Siphoviridae particles, whereas two produced Myoviridae particles. Additionally, pulsed-field gel electrophoresis data indicated that two of the six lysogens were polylysogens, producing more than one distinct type of phage particle. These results suggest that lysogeny is relatively common among soil bacteria.

Host responses influence on the induction of lambda prophage.

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

A novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda.

A novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (UV) induction of bacteriophage lambda in lysogenic Escherichia coli kappa12 (lambda+) has been developed. This method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. In the experiments, 10(8)cells/ml and 30s (39J/m2) were determined as the cell density of the lysogenic bacterium and UV irradiation time, respectively. The reliability of this method was demonstrated by electron paramagnetic resonance (EPR) spectroscopy and spin trapping with DMPO. This method had good reproducibility with intra-day variations (RSD, %) of less than 4% and inter-day variations (RSD, %) of less than 5%, respectively. By this method, the free radical scavenging abilities (ID50) of well-known antioxidants such as glutathione, superoxide dismutase (SOD), catalase (CAT) and carotenoids were determined quantitatively. The results were consistent with the ones obtained by conventional methods for evaluating free radical scavenging abilities. This developed method is reliable and uses common instruments and inexpensive, stable reagents, thus it could be utilized as a routine laboratory quantitative assay to screen a large number of substances that have potential to scavenge the free radical.

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin.

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.

[The phage sensitivity and lysogeny of cultures of Streptococcus pyogenes group A isolated in different streptococcal infections]. / Fagochuvstvitel'nost' i lizogeniia kul'tur Streptococcus pyogenes gruppa A, vydelennykh pri razlichnykh streptokokkovykh infektsiiakh.

The collection of moderate phages of S. pyogenes, group A, had been created earlier. As shown in this work, group A streptococcal cultures isolated from patients with rheumatism, glomerulonephritis and tonsillitis exhibited different sensitivity to the phages of this collection: the cultures were lyzed by phages of groups II and III in rheumatism, group III in tonsillitis and group I in glomerulonephritis. The study revealed that lysogeny was widely spread among S. pyogenes strains isolated from patients with different diseases under study. Most frequently occurred among cultures isolated from tonsillitis patients. In this disease only phage-resistant streptococcal cultures proved to be lysogenic. Lysogeny was found among both phage-sensitive and phage-resistant cultures in rheumatism and especially in glomerulonephritis.

X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio kappa phage.

Vibrio cholerae lysogenic kappa phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated kappa phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m-2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D)--survival (S) curves could be empirically described by the equation S = exp[-(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained.

[Y. pestis phage of a new serovar]. / Fag Y. pestis novogo serovara.

Phage II, isolated from Y. pestis strain 2247 obtained from a desert focus in Central Asia, has been studied. The phage is classified with moderate phages and essentially differs from moderate phages of serovar 2. The sources of isolation, high specificity and the absence of common serological features with presently known Y. pestis phages of serovars 1 and 2 permit the classification of this phage with new serovar 3 of Y. pestis phages.

[The modifying effect of glycerine and cysteamine on prophage lambda induction in E. coli cells under gamma irradiation]. / Modifitsiruiushchee vliianie glitserina i tsisteamina na induktsiiu profaga lambda u kletok E. coli pri gamma-obluchenii.

In studying the modifying influence of glycerol and cysteamine of gamma radiation induction of lambda prophage it was shown that dose curves for lambda prophage induced upon irradiation in normal conditions and in the presence of glycerol are those with a maximum. In the presence of cysteamine, the prophage induction is inhibited significantly.

[Effect of He-Ne laser radiation on the bacteriophage T4-Escherichia coli system]. / Vliianie izlucheniia He-Ne lazera na sistemu bakteriofag T4-Escherichia coli.

Exposure of T4 bacteriophage, having no red light chromophores, to He-Ne laser (lambda = 632.8 nm) of 10(3)-6 X 10(4) J/m2 does not influence its lytic properties. Irradiation of E. coli WP2 bacteria with doses of 4-6 X 10(3) J/m2 causes a 1.25-1.35-fold increase in their ability to keep on the development of nonirradiated bacteriophage T4.

Characterization of Staphylococcus epidermidis mutants sensitive to ultraviolet radiation.

Five UV-sensitive mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphylococcus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr- Umu-; 1 mutant that appears analogous to Uvr-; 1 mutant that resembles LexA- and 1 mutant that exhibits a RecA- phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.

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

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

UV-inducible DNA repair in Acinetobacter calcoaceticus.

Bacterial mutation frequency after UV irradiation and phage mutation frequency under conditions of W-reactivation were determined in A. calcoaceticus. With the exception of streptomycin resistance, there was no increase in the frequency of the assayed markers above the background level. The increased survival of phage during W-reactivation was not followed by an increase in the frequency of mutation from turbid to clear plaque formers among phage survivors. The findings suggested that the UV-inducible repair pathway in A. calcoaceticus was error free. Post-irradiation incubation of UV-treated culture before phage infection resulted in a further increase of W-reactivation. As chloramphenicol inhibited this response, it was concluded that de novo protein synthesis was involved in the UV-inducible repair pathway in A. calcoaceticus.

Prophage induction by ultraviolet light in Acinetobacter calcoaceticus.

UV-induction of prophage P78 of Acinetobacter calcoaceticus increased with the UV-dose given to the lysogenic strain from the spontaneous induction frequency of about 0.8% to a maximal frequency of 10%. This 10- to 20-fold increase of induction frequency, as measured by the number of infective centres, was accompanied by a 1000-fold increase in the yield of free phage. This effect was probably due to an increase in burst size under the conditions of lysogenic induction. Unusually, the lysogen was more resistant to UV-irradiation than the corresponding non-lysogenic strain.

Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1).

Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.

Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12.

A mutation in the purB gene of E. coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30 degrees C. Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work. The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair. Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response. DNA isolated from mud cells is not undermethylated relative to DNA from purB+ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues. Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination. Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.