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PubMed1991

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.

Animals↗

PubMed1991

Compatibility of organic solvents with the Microscreen prophage-induction assay: solvent--mutagen interactions.

The following solvents did not induce prophage lambda in the Escherichia coli WP2s(lambda) Microscreen assay: acetone, benzene, chloroform, ethanol, n-hexane, isopropanol, methanol, toluene, and a mixture of the three isomers of xylene. Dimethyl sulfoxide was genotoxic in the presence and absence of S9, and methylene chloride was weakly genotoxic in the presence of S9. The genotoxic potencies of 2-aminoanthracene and 2-nitrofluorene were reduced when dissolved in DMSO or methanol compared to their potencies when dissolved in acetone.

Anthracenes↗

PubMed1980

Cloning of randomly sheared DNA fragments from a phi 105 lysogen of Bacillus subtilis identification of prophage-containing clones.

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.

Bacillus subtilis↗

PubMed1987

Serotype-converting bacteriophage D3 of Pseudomonas aeruginosa: vegetative and prophage restriction maps.

D3 is a temperate serotype-converting bacteriophage of Pseudomonas aeruginosa. A restriction map, based upon BamHI, PstI, PvuI, HindIII and SmaI sites, indicates that the phage genome is 56.4 kb long, and that it possesses cohesive ends. The prophage map suggests a unique insertion site in the strain AK1380 genome. Phage DNA integration occurs upon the circularization of D3 genome with the integration point approximately equidistant from the two ends.

Bacteriophages↗

PubMed1990

Prophage lambda libraries for isolating cDNA clones by functional screening.

Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque. The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage. The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands. cDNA clones encoding a well-characterized D. discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D. discoideum by functional screening with [125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.

Bacteriophage lambda↗

PubMed1990

Characterisation of a repressor gene (xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX.

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37 degrees C to 48 degrees C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage repressors and suggests that the N-terminal region forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.

Amino Acid Sequence↗

PubMed1998

Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambdaN+cI[Ts]cro- prophage in E. coli cells shifted from 30 degreesC to 42 degreesC, accompanied by loss of immlambda and Rex+ phenotypes and emergence of a non-immune exclusion-state.

The majority of bacteria, which carry the N+-cI857[Ts]-cro--O+-P+ fragment of lambda genome, are killed when derepressed by shifting from 30 degreesC to 42 degreesC. Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical immlambda-immunity phenotype when grown at 30 degreesC; however, when shifted from 30 degreesC to 42 degreesC, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype). We also found that the immlambda survivor cfu quickly lost their Rex+ exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degreesC to 42 degreesC, even though they produced CII, which stimulates pE-cI-rexA-rexB transcription. The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambdawt, and the heteroimmune phage, lambdaimm434. However, lambdavir and spontaneous mutants of lambdawt (lambdase mutations localized within oR) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degreesC. Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes. Phage like lambdawt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny. Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded. The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s). We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degreesC. In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment. The remaining survivor cfu had multiple base substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent. In some of these mutants, either an ochre nonsense mutation or a single-base frameshift deletion inactivated P.

Amino Acid Sequence↗

PubMedDate not supplied

Genome organization in hybrids between prophage phi 80 and Escherichia coli virus phi gamma.

Prophage phi 80 is used for the detection of "discrete" viruses such as phage phi gamma by genetic recombination. Several genetic events have produced a series of hybrids which are currently being characterized. Characteristic genomic properties of the parent phages are recognized in these hybrids. "lambda"-type phages contain a cos-ended DNA of a single sequence, the size of which may be altered by hybridization. "gamma"-type phages contain DNA molecules of uniform size (about 52 kb) and of variable ends; their genome is able to promote highly efficient transduction (pug type) regardless of the origin of the right arm. In both hybrids, the att site is most often a junction point between the parental genomes due to common int-promoted recombination. New evidence is provided for the formation of viable heterozygous "lambda/gamma"-type bacteriophages.

Bacteriophages↗

PubMed1998

A comparative study on the frequency of prophages among natural isolates of Salmonella and Escherichia coli with emphasis on generalized transducers.

Several collections of natural isolates of the genus Salmonella and of the species Escherichia coli were studied for the release of viable temperate phages. The results indicated that functional prophage genomes may be a common constituent of all bacterial genomes of the investigated strains. About 99% of the Salmonella phages are capable of generalized transduction of chromosomal host markers and plasmids. The ratio of transducing E. coli phages is significantly lower.

Animals↗

PubMedDate not supplied

The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage.

Toxic shock syndrome (TSS) is a complex of generalized symptoms caused by a local staphylococcal infection, and a circulating toxin is thought to be involved. Indeed, nearly 100% of TSS isolates produce an exoprotein, TSSE, that is thought to have an aetiological role on the basis of positive animal tests (refs 1,2 and F. Quimby, personal communication) and human serological data. Although the precise role of TSSE in TSS remains unclear (E. Kass, personal communication), no other staphylococcal factor has been implicated. Our preliminary studies of the genetics of TSSE production failed to demonstrate plasmid or phage involvement or linkage with known chromosomal genes (ref. 4 and B.N.K. et al., unpublished data); however, Schutzer et al. have found that most TSS strains harbour prophages with common plating characteristics and suggest that the toxin(s) involved in TSS are transmitted by lysogenic conversion. We show here that TSSE is not demonstrably transferred by lysogeny; moreover, we have cloned the gene and found that the cloned product is serologically and biologically indistinguishable from the native protein, and that the TSSE determinant is associated with a larger DNA segment that is absent or rearranged in TSSE- strains.

Animals↗

PubMed2001

The tRNA function of SsrA contributes to controlling repression of bacteriophage Mu prophage.

The small regulatory RNA SsrA has both tRNA and mRNA activities. It charges alanine and interacts with stalled ribosomes, allowing for translation to resume on the SsrA mRNA moiety. Hence, unfinished peptides carry a short amino acid tag, which serves as a signal for degradation by energy-dependent proteases. In SsrA-defective Escherichia coli strains, thermoinducible mutants of the transposable bacteriophage Mu (Mucts) are no longer induced at high temperature. Here we show that truncated forms of the key regulator of Mu lysogeny, the repressor Repc, accumulate in the absence of SsrA. These forms resemble C-terminally truncated dominant Mu repressor mutants previously isolated from Mucts, which are no longer thermoinducible and bind operator DNA with a high affinity even at high temperature. Using various ssrA alleles, we demonstrate the importance of SsrA charging on the ribosome for controlling Mu prophage repression. Our results thus substantiate the previous observation that trans-translation is not the only function of the SsrA. The alternative function of SsrA appears to influence the stability of Mu lysogens by controlling the translation of the C-terminal domain of the repressor protein, which modulates the affinity of the protein for DNA and its susceptibility to proteolytic degradation.

Alanine↗

PubMed1969

The molecular origin of lambda prophage mRNA.

Lambda-specific RNA extracted from lysogenic bacteria hybridizes specifically with fragments of lambda DNA containing 43 per cent GC (guanine plus cytosine). Therefore genes known to function in the prophage state (c(I) and rex) lie 0.38 +/- 0.08 fractional molecular length from the right end of the lambda DNA molecule, according to the compositional map of Skalka, Burgi, and Hershey.

Centrifugation, Density Gradient↗

PubMed1975

Excision of prophage lambda in a cell-free system.

A cell-free system that promotes the excision of prophage lambda DNA has been established. The substrate for the reaction is phage DNA carrying two attachment sites, which, in vivo, undergoes intramolecular recombination between these sites. The in vitro recombination system is efficient; 25-35% of the substrate DNA undergoes recombination in 30 min. There is an absolute requirement for ATP; Mg++ and spermidine are stimulatory. RNA does not appear to be involved, nor can a role for DNA synthesis be demonstrated.

Adenosine Triphosphate↗

PubMed1977

Initiation of genetic exchanges in lambda phage--prophage crosses.

When Escherichia coli K-12 (lambda) lysogens were infected with lambda phages, genetic exchanges between phage and prophage occurred at low frequencies (less than 0.1% between the markers P3 and P80), but at frequencies above 1% if the infecting phages were first treated with the photosensitizing agent 4,5',8-trimethylpsoralen and 360 nm light. Exchanges were induced by psoralen damage at about the same frequency in wild-type lysogens and in those carrying recB(-), recC(-), recF(-), or lexA(-), but at an intermediate frequency in a quadruple mutant carrying recB(-)recC(-)recF(-)sbcB(-). Few if any exchanges were induced in lysogens carrying uvrA(-), uvrB(-), or recA(-). The increase in the frequency of recombination was presumably due to the psoralen damage in the phage DNA molecules and the action of host cell repair and recombination enzymes. The production of crosslinks in the phage DNA by psoralen and 360 nm light was measured by sedimentation in alkali. It showed second-order kinetics indicative of a two-photon reaction. In contrast, first-order kinetics had been reported for monoadduct formation. Second-order kinetics, similar to those for crosslink production, were found for genetic exchanges in homoimmune crosses. Presumably, crosslinks, rather than monoadducts, cause most of the exchanges. Because the uvrA(-) gene product (UV-endonuclease) was required, it is likely that recombination was initiated by DNA molecules cut at crosslinks. This system has been used to show that after the crosslinked phage duplex has been cut, one or more of the subsequent steps-homologous pairing, cutting, and joining-require the recA(+) gene product.

Coliphages↗

PubMed1989

Activation of prophage P4 by the P2 Cox protein and the sites of action of the Cox protein on the two phage genomes.

Phage P2 induces the unrelated prophage P4. In this paper we show that this is due to the activation of the P4 late promoter PII by the P2 Cox protein. This is in contrast to the effects of Cox on P2, for which it is known from previous work that it acts as a repressor of the promoter Pc, which is responsible for expression of the immunity repressor C. The activator role of Cox was revealed by its effect on replication of P4 DNA and on the formation of chloramphenicol acetyltransferase when a promoterless cat gene was inserted downstream of the P4 PII promoter. DNase I protection studies revealed that the Cox protein binds to the repressor promoter Pc of phage P2 and to the promoter PII of phage P4. In the latter case the Cox protein binds upstream of the -35 region, in analogy to several other activators of promoters. A weak binding was found in the promoters Pe of phage P2 and Ple of phage P4. The Cox protein is a case of viral transactivation of the replication genes of one phage by a control protein of the other. However, the effects of the Cox protein are totally different in the two phages, repressive in one case and activating in the other.

Base Sequence↗

PubMed1975

Prophage inductive efficiency of alkylating agents and radiations.

The prophage inducing efficiency in E. coli K-12 (lambda) of a number of agents--alkylating agents and radiations--has been compared at a high survival of bacteria. The inducing effectiveness (per alkylation in DNA) and efficiency (compared with mutation frequency in E. coli Sd-4) was found to decrease in the order 2-hydroxyethylating agents (2-hydroxyethyl methanesulphonate and ethylene oxide) greater than isopropyl methanesulphonate approximately equal to methyl methanesulphonate greater than ethylating agents (diethyl sulphate, ethyl methanesulphonate). The low inducing activity of the ethylating agents could not be explained with respect to their reactivity towards targets of differing nucleophilicity, nor could the effectiveness in mutagenicity or the ability to break the chromosomes in the presence of metal ions be invoked. The high inducing efficiency of hydroxyalkylating agents may be related to their ability to break DNA strands.

Alkylating Agents↗

PubMed1991

Lysogeny of methicillin-resistant Staphylococcus aureus and the role of prophages in transfer of conjugative and non-conjugative plasmids.

The lysogenicity of 49 strains of methicillin-resistant S. aureus (MRSA) isolated in Moscow clinics in the 1970s and '80s was studied by the method of mitomycin C induction. It was found that one strain had phage of serogroup B, 33 strains had serogroup F phages and 15 strains had phages of both serogroups. In the course of genetic crossing on nitrocellulose filters it was demonstrated that serogroups B and F prophages contained in recipient cells 1) increase the frequency of transfer of conjugative plasmid pG873 and 2) mobilize transfer of non-conjugative plasmids pE994 and rms7.

Conjugation, Genetic↗

PubMed1987

A novel P22 prophage in Salmonella typhimurium.

Under several sets of conditions, all of which seem to perturb purine metabolism, Salmonella typhimurium releases a variety of phages which were not known to be present in the strain. These cryptic phages are not induced by UV irradiation. Furthermore, the induction process does not require a functional recA gene product. While phages of several phenotypic classes have been recovered, including both turbid and clear plaque formers, all appear to be variants of P22 because all show DNA restriction patterns indistinguishable from that of P22. The variety of types suggests that the cryptic prophage is mutagenized as a consequence of the induction process. All the temperature phages tested are capable of transducing a variety of chromosomal markers with high efficiency. The phages induced in this novel way are capable of forming plaques on the strains that gave rise to them. Since the strains releasing phage are not immune to P22, the parental lysogens must not express immunity and the phage must be held in a cryptic state by a novel mechanism. The released phage possess an intact P22 immunity system because many can form standard immune lysogens after reinfection of Salmonella. These results raise the possibility that Salmonella typhimurium harbors cryptic phages that are subject to a novel system of global control related to purine metabolism. Preliminary evidence suggests that the regulation system may involve DNA modification.

Anaerobiosis↗