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PubMed1975

The kinetics of derepression of prophage lambda following ultraviolet irradiation of lysogenic cells.

Double lysogens for prophages lambda cI+ and lambda cI ind-ts-857 are induced only by the combined effects of ultraviolet (UV) irradiation and high temperature, not by either treatment alone (Sussman and Jacob, 1962). We have followed the kinetics of inactivation of the cI+ repressor brought about by irradiation in asynchronously and synchronously growing cultures of B/r (lambda cI ind- ts-857). Assays of the yield of phage released as a result of temporary thermal inactivation of the UV-resistant ind- ts-857 repressor at intervals after the irradiation accurately reflect the time course of UV-induced inactivation of the cI+ repressor. The results show that UV-induced derepression takes place in all cells of the population approximately 20 min after the irradiation whether the cells were growing asynchronously or synchronously. Hence UV induction of prophage lambda is not triggered at a particular stage in the cell cycle.

Cell Count↗
PubMed1976

Insertion sequence IS2 near the gene for prophage lambda excision.

In this study we characterize a variant of the lambdacI857S7 prophage, designated lambdabi2cI857S7, which carries a DNA insertion. The insertion sequence is IS2, and it resides in the antipolar orientation II just upstream from the gene for prophage excision (xis) at 61.6%lambda. This bi2 insertion mutant could prove valuable for studies on possible recombination functions of IS2 DNA and of its effect on the lambda integration and excision functions.

Chromosome Mapping↗
PubMed1975

Compatibility properties of P1 and PhiAMP prophages.

Although phages P1 and PhiAmp are heteroimmune (Chesney and Scott, 1975 and Yarmolinsky, unpublished), their plasmid prophages are incompatible. Thus, the immunity and compatibility systems are two distinct regulators of phage replication. The two prophages, and plasmid P15B (Ikeda, Inzuka and Tomizawa, 1970) constitute a new compatibility group, designated Y.

Centrifugation, Density Gradient↗
PubMed1980

Effects of prophage Mu induction on expression of adjacent host genes.

The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of beta-lactamase. The expression of the lacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.

Bacteriophage mu↗
PubMed1995

Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

Bacillus subtilis↗
PubMed1983

Induction of prophage lambda in Escherichia coli recA- strain by N-methyl-N'-nitro-N-nitrosoguanidine.

Induction of prophage by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) occurred in a recA- strain lysogenic for lambda phage at a level significantly higher than the spontaneous level although the frequency was much lower than that of induction in a recA+(lambda) strain. The plaque-forming ability of lambda c17 super-infecting the recA-(lambda) strain pretreated with MNNG increased with dose of MNNG as it did for super-infection of the recA+(lambda) strain, indicating that the frequency of maturation of lambda c17 increased owing to a decrease in the immunity of the lambda lysogen with dose of MNNG given to it. Further, the activity of lambda repressor in the recA-(lambda) strain decreased after treatment with MNNG as measured by the decrease of repressor-binding activity to lambda DNA although it decreased at a 3-fold slower rate than that in recA+(lambda) strain. From these results and others previously reported we conclude that inactivation of repressor leading to MNNG-initiated prophage induction takes place through two pathways, one being the recA-dependent normal process and the other a recA-independent process unique to the effect of MNNG.

Bacterial Proteins↗
PubMed1984

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents.

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.

Bacteriophages↗
PubMed1987

Relative mutagenic and prophage-inducing effects of mono- and di-alkyl nitrosoureas.

Mutagenic capacity, prophage-inducing ability, and decomposition rates of mono- and di-alkylnitrosoureas with the same alkyl groups were studied in aqueous systems using S. typhimurium strain TA1535 and E. coli strain BR339 (lambda). Slower decomposition rates of nitrosodialkylureas compared with monoalkylnitrosoureas were not reflected in reduced mutagenicity, with the exception of the methylating agents. With the monoalkylnitrosoureas, mutagenicity varied with length of the alkyl chain, and was greatly increased by oxygen substituents. Among the dialkylnitrosoureas, mutagenesis and decomposition rates were dependent on the substitutent in the N-1 position, adjacent to the nitroso group. Prophage induction, on the other hand, was dependent on a substituent in the N-3 position. The results suggested the existence of two distinct mechanisms for DNA damage, one SOS-dependent and the other SOS-independent, by which different dialkylnitrosoureas acted. Neither in vitro assay system was a reasonable model for the carcinogenic activity of these compounds.

Alkylation↗
PubMed1993

Enhancement of the prophagic but not of the antidipsogenic effect of U-50, 488H after chronic amphetamine.

Two groups of rats were treated with seven daily injections of either saline or d-amphetamine (3 mg/kg IP). On the 2 days following the last injection, rats were tested according to a counterbalanced experimental design, each animal receiving, immediately prior to the beginning of the dark phase, saline on one day and the highly selective kappa-opioid agonist trans- +/- 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzene- acetamide methanesulfonate hydrate [U-50,488H (U50)] on the other. A microcomputer-controlled data acquisition system was used for the structural analysis of the feeding and drinking responses to amphetamine and U50. U50 enhanced feeding and depressed drinking in the first hour. The increased food intake was probably the result of the effect of U50 on the development of satiation and duration of satiety. Chronic amphetamine potentiated the prophagic effect but not the antidipsogenic effect of U50. The structural analysis demonstrated that the characteristics of the prophagic effect of U50 were amplified but not changed.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1983

Role of the Xis protein of bacteriophage lambda in a specific reactive complex at the attR prophage attachment site.

Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions. The lambda Int protein is required for both directions, but Xis for excision only. Previous electron microscopic observations have shown that Int protein forms a stable, condensed protein-DNA complex with the phage (attP) and prophage left (attL) substrate sites, but not with the host (attB) or prophage right (attR) sites. We have found that Int and Xis together produce a stable, condensed complex with attR. The attR complex involves the P region DNA to the left of the crossover point (O site). In contrast, the attP complex includes DNA on both sides of the crossover point (P and P'), and the attL structure involves the P' DNA to the right of O. In the presence of Int and Xis, the attL and attR sites form a paired structure. We conclude that the role of Xis is to provide a distinct reactive structure at attR, allowing attL and attR to pair efficiently.

Bacteriophage lambda↗
PubMed1986

Micro-BIA, a colorimetric microtiter assay of lambda prophage induction.

Escherichia coli that are lysogenic for a lambda-lacZ fusion phage produce beta-galactosidase, product of the lacZ gene, upon induction of the prophage by DNA-damaging agents. The miniaturization of a quantitative, colorimetric beta-galactosidase (prophage) induction assay (BIA) is presented. Induction assays are performed in microtiter wells with the aid of multichannel pipetting devices. Results are shown with screening strain BR513 (uvrB delta envA) and a strain, BR339 (uvrB delta lexA3ind-) which exhibits enhanced induction. A method developed for strain BR339 utilizes bacteria stored frozen in log phase, permeabilized in vitro, and used immediately; with this method, 2 consecutive assays may be completed in 1 working day. Mutagens utilized for the model studies included 4NQO, ENNG, daunorubicin, bleomycin, acetoxy-AAF, B[a]P, DMBA, and DEN (the last three in the presence of liver S9). Induced levels of beta-galactosidase were monitored using a vertical light path photometer that measured color absorbance in each microtiter well. Alternatively, color intensity could be determined by using a color chart prepared for this assay. These values were then plotted to generate dose-response curves. Considerable savings in labor and materials are achieved with the method described, one which may be used as a screen for DNA-damaging chemicals. Automated equipment and computers may be used to advantage with this assay, but they are not required.

Animals↗
PubMed1983

Roasting coffee beans produces compounds that induce prophage lambda in E. coli and are mutagenic in E. coli and S. typhimurium.

Freshly brewed blended coffee, instant coffee and instant caffeine-free coffee induced prophage lambda in lysogenic E. coli K12, strain GY5027. Because coffee prepared from green beans by the same extraction method as used for freshly brewed blended coffee had no prophage-inducing activity, this activity may be attributed to compounds produced in the roasting process. Roasting also produced compounds that were mutagenic in S. typhimurium TA100 and E. coli WP2 uvrA/pKM101.

Bacteriophage lambda↗
PubMed1983

Repression of UV induction of lambda prophage by caffeine.

Caffeine was found to function as a repressor of UV-induced lambda prophage when added to the post-irradiation culture medium with both uvrB+ and uvrB- strains of E. coli K12. Caffeine functioned as a repressor of lambda induction at high but not low inducing doses of UV. Caffeine alone at concentrations above 10 mg/plate functioned as an inducer of lambda prophage.

Bacteriophage lambda↗
PubMed1990

DNA damage and prophage induction and toxicity of nitrofurantoin in Escherichia coli and Vibrio cholerae cells.

Repair-deficient and repair-proficient strains of E. coli K12 were sensitive to nitrofurantoin treatment to varying degrees with the double mutant strain (uvrA 6, recA 13) being most sensitive. Ultraviolet absorption data and thermal chromatography through a hydroxyapatite column revealed that nitrofurantoin treatment of V. cholerae strain OGAWA 154 produced a maximal amount of 55% reversibly bihelical DNA at a nitrofurantoin dose of 120 micrograms/ml/h, which indicated the formation of inter-strand cross-links in DNA. Nitrofurantoin also produced prophage-lambda induction in E. coli K12 strain GY 5027: envA, uvrB, ampA 1, strA (lambda), in a dose-dependent manner, the maximum induction being highly significant (P less than 0.001). Previously published mutation data coupled with the prophage induction data presented here suggest that the genotoxic properties of nitrofurantoin are mediated through the SOS pathway.

Bacteriophage lambda↗
PubMed1982

Isolation and characterization of Streptomyces fradiae plasmids which are prophage of the actinophage phiSF1.

Two plasmids, designated pUC1 and pUC13, have identified as different prophage states of the Streptomyces fradiae actinophage phiSF1. Plasmids pUC1 and pUC13, 51.1 Md, are identical as determined by restriction endonuclease analysis and other criteria. They differ in that while pUC1 specifies "type A" plaques, characterized by a narrow zone of growth inhibition around a transformant colony, pUC13 specifies "type B" plaques, characterized by concentric rings of growth and growth inhibition around a transformant colony. Additionally cultures carrying pUC13 release antinophage phiSF1 at a frequency about 10 3-10 4 times that pUC1 containing cultures. Restriction enzyme digestion, transfection, and transformation analyses show that phiSF1 DNA, 55.7 Md, is about 8-10% larger than pUC13 and appears to be circularly permuted and terminally redundant. The pUC1 and pUC13 prophage are interconvertible.

Bacteriophages↗
PubMed2004

Bacteriophage P4 Vis protein is needed for prophage excision.

Upon infection of its host Escherichia coli, satellite bacteriophage P4 can integrate its genome into the bacterial chromosome by Int-mediated site-specific recombination between the attP and the attB sites. The opposite event, excision, may either occur spontaneously or be induced by a superinfecting P2 helper phage. In this work, we demonstrate that the product of the P4 vis gene, a regulator of the P4 late promoters P(LL) and P(sid), is needed for prophage excision. This conclusion is supported by the following evidence: (i) P4 mutants carrying either a frameshift mutation or a deletion of the vis gene were unable to excise both spontaneously or upon P2 phage superinfection; (ii) expression of the Vis protein from a plasmid induced P4 prophage excision; (iii) excision depended on a functional integrase (Int) protein, thus suggesting that Vis is involved in the formation of the excision complex, rather than in the excision recombination event per se; (iv) Vis protein bound P4 DNA in the attP region at two distinct boxes (Box I and Box II), located between the int gene and the attP core region, and caused bending of the bound DNA. Furthermore, we mapped by primer extension the 5' end of the int transcript and found that ectopic expression of Vis reduced its signal intensity, suggesting that Vis is also involved in negative regulation of the int promoter.

Amino Acid Sequence↗
PubMed2004

Diversity in the lysis-integration region of oenophage genomes and evidence for multiple tRNA loci, as targets for prophage integration in Oenococcus oeni.

The central genomic regions of Oenococcus oeni phages fOg30 and fOgPSU1 have been compared with the equivalent regions of oenophages fOg44 and phi 10MC. In all cases, an almost identical endolysin gene was followed by one of two orfs, encoding putative holins (orf117 and orf163). The fOg44 endolysin was established as a secretory protein when expressed in Lactococcus lactis. Orf117 (from fOg44) promoted lysis of Escherichia coli cultures upon induction of a defective lambda Sam7 prophage, but Orf163 (from fOg30) failed to elicit a lysis response in this system. fOg44 and fOgPSU1 were shown to integrate at the 3' end of a tRNA(Glu) and a tRNA(Lys), respectively. Searching the available sequence of the O. oeni MCW genome for attP-like elements, two other tRNA targets could be proposed for prophage establishment. Between the lysis and integration elements, a diverse cluster of genes (absent in phi 10MC) was observed. One common gene in this "lysogenic conversion cluster" was experimentally confirmed as a transcriptional repressor, affecting the expression of a putative permease gene.

Amino Acid Sequence↗