Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle.

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.     

Induction of Prophage SPO2 in Bacillus subtilis by 6-(para)-Hydroxyphenylazouracil

DNA replication in Bacillus subtilis is reversibly inhibited by 6-(para)-hydroxyphenylazouracil (HpUra) (1971), whereas replication of temperature phage SPO2 is not affected by the drug. Experiments are presented which show that HpUra will induce bacteria lysogenic for SPO2. Also, prophage phi105, which is sensitive to the drug, is induced by HpUra. Induction of SPO2 lysogenics in the presence of HpUra gives selective synthesis of SPO2 DNA.     

Deoxyribonucleic Acid Polymerase Activity in a Deoxyribonucleic Acid Polymerase I-Deficient Mutant of Bacillus subtilis Infected with Temperate Bacteriophage SPO2

Increased deoxyribonucleic acid (DNA) polymerase activity is found in soluble extracts from a polymerase I-negative mutant of Bacillus subtilis after infection with temperate phage SPO2, or after induction of SPO2 prophage in lysogenic derivatives of this mutant. No increased enzyme activity is found after SPO2 infection in the presence of chloramphenicol. Infection of the polymerase-negative mutant with the DNA-negative sus mutant SPO2 L244 gives no increased enzyme activity, whereas infection with DNA-negative sus mutant SPO2 J385 gives enzyme activities comparable to those found in wild-type infected cells. These findings suggest that SPO2 determines a DNA polymerase activity essential for synthesis of phage DNA.     

Prophage Induction of Noninducible Coliphage 186

Coliphage 186 has been regarded as a member of the noninducible group of coliphages. Evidence that prophage 186 is induced by ultraviolet irradiation or by treatment with nalidixic acid or mitomycin C is now presented. The phage yields were similar to those from lysogens of the inducible phage lambda, and the induction required a recA(+) host. A noninducible mutant of 186 was isolated from its heat-inducible derivative, 186cIts, that was no longer inducible by ultraviolet irradiation but remained heat inducible. That zygotic induction of 186 after transfer from a lysogenic male to a non-lysogenic recipient did not occur is indicated by the following findings: (i) there was only a slight increase in phage titer; (ii) similar levels of recombinants were obtained for markers adjacent or distal to the phage integration site, whether the recipient was lysogenic or not, and there was no effect on the gradient of marker transfer; (iii) lysogenic recombinants were readily found and the co-transfer of 186 with adjacent markers was the same to lysogenic or non-lysogenic recipients. Thus, 186 formed an inducible prophage that did not display zygotic induction. Nevertheless, it shared many properties with the noninducible phage P2 as outlined in the discussion.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

Prophage Curing in Lactobacillus casei by Isolation of a Thermoinducible Mutant

To eliminate the occurrence of virulent phage in industrial fermentation, attempts were made to obtain prophage-cured derivatives from Lactobacillus casei lysogenic strain S-1. A thermoinducible mutant lysogen was isolated from mutagenized strain S-1, since S-1 cannot be induced under laboratory conditions. The mutation responsible for thermoinducibility was located on the prophage. Prophage-cured strains were selected after heat induction of the mutant. These cured strains did not produce the virulent phage and should be valuable for industrial fermentation.     

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.     

Relationship Between Lysogeny, Spontaneous Induction, and Transformation Efficiencies in Bacillus subtilis

The low transformation efficiency of Bacillus subtilis 168 lysogenic for phages ?105 or SPO2 is shown to result from the induction of lytic phage replication in competent cells. Lysogenic competent cells have a higher rate of spontaneous prophage induction than noncompetent cells. Mutants of ?105 and SPO2 which form lysogens resistant to spontaneous induction were isolated, and these lysogens exhibited higher transformation levels than those formed by wild-type phage. These results suggest that the physiological state of competence in B. subtilis promotes prophage derepression leading to cell death and the loss of potential transformants.     

Prophage Induction by High Temperature in Thermosensitive dna Mutants Lysogenic for Bacteriophage Lambda

High-temperature treatment of thermosensitive dna mutants lysogenic for phage lambda leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes dnaB, dnaE, and dnaG. In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all dnaB(lambda) mutants tested, but not in mutants of the genotypes dnaE(lambda) and dnaG(lambda). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.     

Prophage contribution to bacterial population dynamics

Cocultures of Salmonella strains carrying or lacking specific prophages undergo swift composition changes as a result of phage-mediated killing of sensitive bacteria and lysogenic conversion of survivors. Thus, spontaneous prophage induction in a few lysogenic cells enhances the competitive fitness of the lysogen population as a whole, setting a selection regime that forces maintenance and spread of viral DNA. This is likely to account for the profusion of prophage sequences in bacterial genomes and may contribute to the evolutionary success of certain phylogenetic lineages.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Linked Transformation of Bacterial and Prophage Markers in Bacillus subtilis 168 Lysogenic for Bacteriophage φ105

Level of competence reached by Bacillus subtilis 168 lysogenic for temperate phage phi 105 was reduced compared to that reached by nonlysogenic cells. This effect was probably related to an alteration of the bacterial surface. Deoxyribonucleic acid extracted from phi 105 lysogenic bacteria was used to transform other lysogenic bacteria. About 25% linkage was found between the bacterial phe-1 marker and prophage marker ts N15. The order of a few prophage markers relative to phe-1 was established in three-factor crosses. The usefulness of this system for a study of the linkage between an integrated prophage genome and that of its host was discussed.     

Defective Bacteriophage PBSH in Bacillus subtilis: II. Intracellular Development of the Induced Prophage

Treatment of Bacillus subtilis strain 168 with mitomycin C caused induction of a defective prophage, PBSH. During induction, extensive deoxyribonucleic acid (DNA) synthesis took place. Concurrently, a change in marker frequency of the bacterial DNA was noticed. The frequency of only one marker, ade-16, the marker closest to the origin of the bacterial chromosome, was enhanced manyfold. DNA from whole phage particles transformed all bacterial markers at a frequency equal to that of DNA in the noninduced culture, except ade-16, the frequency of which was enhanced 30 to 100 times. Analysis of a double isotope experiment demonstrated that 14% of the phage DNA was derived from preinduction bacterial DNA. The other 86% of DNA in phage particles was DNA replicated after induction. Density label experiments with 5-bromodeoxyuridine showed that postinduction DNA synthesis took place preferentially at the origin region of the bacterial chromosome. Measurement of the molecular weight of DNA replicated after induction clearly showed that postinduction DNA replication is chromosomal. No evidence for prophage detachment and autonomous phage DNA replication was found. The data indicated that, after mitomycin C action, the bacterial chromosome under-went multiple reinitiation at the origin, while normal sequential DNA replication was stopped. The pool of replicated bacterial DNA was fragmented randomly. This DNA was packaged into PBSH particles which were released after cell lysis.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.     

Seasonal Variation in Lysogeny as Depicted by Prophage Induction in Tampa Bay, Florida

A seasonal study of the distribution of lysogenic bacteria in Tampa Bay, Florida, was conducted over a 13-month period. Biweekly water samples were collected and either were left unaltered or had the viral population reduced by filtration (pore size, 0.2 μm) and resuspension in filtered (pore size, 0.2 μm) water. Virus-reduced and unaltered samples were then treated by adding mitomycin C (0.5 μg ml⁻¹) to induce prophage or were left untreated. In order to test the hypothesis that prophage induction was phosphate limited, additional induction experiments were performed in the presence and absence of phosphate. Induction was assessed as an increase in viral direct counts, relative to those obtained in controls, as detected by epifluorescence microscopy. Induction of prophage was observed in 5 of 25 (20%) unaltered samples which were obtained during or after the month of February, paralleling the results from a previous seasonal study. Induction of prophage was observed in 9 of 25 (36%) of the virus-reduced samples, primarily those obtained in the winter months, which was not observed in a prior seasonal study (P. K. Cochran and J. H. Paul, Appl. Environ. Microbiol. 64:2308-2312, 1998). Induction was noted in the months of lowest bacterial and primary production, suggesting that lysogeny was favored under conditions of poor host growth. Phosphate addition enabled prophage induction in two of nine (22%) experiments. These results indicate that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration, suggesting that phosphate concentration may modulate the lysogenic response of natural populations.     

DNA Replication of Induced Prophage in Haemophilus influenzae

DNA synthesis during transition from the lysogenic state to the lytic cycle and throughout the latter has been studied in Haemophilus influenzae BC200 (HP1c1). Following exposure to ultraviolet light, there is a 30-min delay in DNA synthesis after which there is a rapidly increasing rate of phage DNA synthesis. The phage genome is replicated without extensive utilization of segments or of breakdown products of the bacterial chromosome. The mode of phage DNA replication was investigated by zonal sedimentation of labeled DNA in 5 to 20% neutral and alkaline sucrose gradients. Tritiated thymidine, incorporated during a 2-min pulse given at 38 min, chases rapidly into DNA, sedimenting like linear DNA of approximately 2 x 10(8) daltons, and then, at the expense of label in this peak, chases into slower-sedimenting phage DNA (2 x 10(7) daltons). The fast-sedimenting, rapidly labeled DNA satisfies certain criteria for being a concatenated replicative intermediate. Observations in the electron microscope revealed linear concatemers in the faster-sedimenting material and circular phage-sized DNA in the slower-sedimenting DNA. When induced cells are gently lysed with lysozyme and Brij 58 to maintain DNA-membrane associations and sedimented in neutral sucrose over a cesium chloride shelf, the concatemer is found with the cell-membrane-wall complex. Membrane-associated label chases to membrane-free material sedimenting like deproteinized HP1c1 DNA. When membrane-associated DNA from the cesium chloride shelf is deproteinized and resedimented in neutral sucrose, the sedimentation profile reveals that sedimentation rates of labeled DNA from this complex are indicative of sizes ranging from 2 x 10(8) daltons down to phage-sized pieces of 2 to 3 x 10(7) daltons. A model is presented which places HP1c1-DNA replication on the cell membrane where a concatemer of phage DNA is synthesized and subsequently degraded to phage-equivalent DNA. Phage-equivalent DNA is then either released from the membrane for packaging or is packaged while still membrane associated. Thus, the cell membrane is not only the site of DNA replication during which phage DNA is synthesized in multiple phage-equivalent concatemers but it is also the site at which these concatemers are selectively reduced to phage-sized pieces.