Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Microbial short-term assays with thiram in vitro

The fungicide thiram was assayed in the following tests in vitro, with and without metabolic activation: (1) prophage lambda induction of Escherichia coli K12; (2) repair test in Salmonella typhimurium (strains TA1538 and TA1978); (3) induction of gene mutations in Aspergillus nidulans (methA1 suppressor induction). Thiram was positive in the repair test and in the A. nidulans forward-mutation test (4-6 fold increase) in the absence of metabolic activation. A slight increase was observed in prophage lambda induction with thiram in the presence of the metabolic activation system.     

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.     

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.     

Cell Division and Prophage Induction in Escherichia coli: Studies of Nucleotide Levels

Cell division and prophage repression in the Escherichia coli mutant, T-44, are very sensitive to the levels of certain purine and pyrimidine derivatives in the media. The hypothesis that a change in the level of an adenine derivative in the small molecule pool of this strain was responsible for prophage induction and filament formation was tested. The nucleoside triphosphate pools in T-44 and C-600 nonlysogenic and lysogenic strains were labeled in experiments with (32)P and (33)P. Cultures were mixed, and the nucleotides were isolated. When adenine was present, the level of adenosine triphosphate (ATP) in T-44 compared to C-600 (as indicated by the isotope ratio) was increased up to twofold. Most of the other nucleotides increased but not to the same degree. In the lysogenic strain guanosine triphosphate and deoxycytidine triphosphate showed increases comparable to ATP, whereas increases noted in the deoxynucleotides in T-44 +/- lambda with adenine present were less. In experiments where T-44 and C-600 were incubated with (3)H- and (14)C-adenine, the levels of several compounds, including ATP, were slightly elevated in T-44. The combined data suggest that cultures of T-44 +/- lambda, grown in the presence of adenine, show a preferential increase in the level of ATP when compared to C-600 +/- lambda, but the increase in relation to the other nucleotides is less than twofold. In the experiment with (3)H- and (14)C-adenine, the level of inosine was found to be increased in T-44 relative to C-600. Cyclic AMP, when added to cultures of T-44 under various conditions, had no effect on prophage induction. Intracellular and extracellular levels of cyclic AMP in T-44 compared to C-600, incubated with had-acidin, guanosine, and cytidine (HGC) or with HGC plus adenine, were not significantly different. No compelling evidence for altered nucleotide metabolism in T-44 +/- lambda as a cause of prophage induction or filament formation was obtained.     

Effect of glutathione on UV induction of prophage lambda

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m⁻²), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.     

Lambda excision revisited: testing a model for synapsis of prophage ends

Excision of lambda prophage was reexamined to test a model for prophage end synapsis. The model proposes that, during in situ prophage replication, following induction, the diverging replication forks are held together. Consequently, prophage DNA is spooled through the replication machinery, drawing the prophage ends together and facilitating synapsis. The model predicts that excision will be slowed if in situ lambda replication is inhibited, and the predicted low rate of excision of a nonreplicating prophage was observed after thermoinduction. However, excision was rapid if additional Int protein was supplied or if the temperature was reduced after induction, showing that (i) Int is partially thermosensitive for excision at 42 degrees C and (ii) in situ replication is not required for rapid excision, a finding that is inconsistent with the model.     

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.     

The modifying effect of glycerine and cysteamine on prophage lambda induction in E. coli cells under gamma irradiation

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.     

Induction of prophage lambda by daunorubicin and derivatives correlation with antineoplastic activity

The antineoplastic drug daunorubicin and 15 other anthracyclines were tested for their ability to induce prophage lambda in Escherichia coli K12. Prophage lambda induction by daunorubicin was obtained in excision-repair deficient uvr- bacteria at doses about 3-fold lower than in excision-repair proficient uvr+ cells; this suggests that some of the lesions produced in DNA by daunorubicin are subject to excision repair and may be adducts. Daunorubicin seems to be converted to active species capable of causing prophage inducing lesions in DNA by bacterial enzymes. The antineoplastic and prophage inducing potencies of the anthracyclines were compared in a blind test. These two parameters were correlated for two thirds of the compounds. Such a correlation supports the idea that the antineoplastic activity of the anthracyclines is a consequence of their capacity to damage DNA.     

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.     

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.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Determinants of segregational stability of the linear plasmid-prophage N15 of Escherichia coli

N15 is a bacteriophage of Escherichia coli that resembles lambda, but, unlike lambda, it lysogenizes as a linear plasmid. We show that stable maintenance of this unusual plasmid-prophage depends on the parA and parB genes, relatives of the partition genes of F and P1 plasmids. ParB of N15, like its F- and P1-encoded homologues, destabilizes plasmids carrying its target centromere, when present in excess. Within the genome of N15, we identified four unlinked, palindromic sequences that can promote the ParB-mediated destabilization of a moderate-copy vector in cis. They are distant from the parAB operon, unlike the centromeric sites of F and P1. Each of these palindromes could interact in vivo and in vitro with ParB. Each, when cloned separately, had properties characteristic of centromeric sites: exerted incompatibility against the N15 prophage and mini-N15 plasmids, and stabilized a mini-P1 plasmid depleted of its own partition genes when ParA and ParB of N15 were supplied. A pair of sites was more effective than a single site. Two of the centromeric sites are located in the proximity of promoters of phage genes, suggesting that, in addition to their function in partitioning of N15 prophage, they may control expression of N15 lytic functions.     

The mechanisms of the manifestation of the genotoxic effects of binase]

The high concentrations of commercial sample of Bacillus intermedius 7P ribonuclease (binase) showed a weak mutagenic effect in Ames test and Ara-test. It has been established that binase induces the SOS-functions of microbial cell and prophage induction. The way of exogenic enzyme action through activation of RecA protein is proposed.