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.     

Temperate phage induction and filament formation in Vibrio cholerae by furazolidone

Furazolidone a synthetic nitrofuran used in the treatment of cholera, was found to cause prophage induction and filament formation in Vibrio cholerae. Maximum induction of phage beta was obtained at a drug concentration of 0.5 microgram/ml. Neither induction of prophage nor filamentation took place if the drug treatment was carried out in the presence of 2.5 micrograms/ml chloramphenicol, indicating a requirement for de novo protein synthesis. Our results strongly suggest the existence of "SOS" type functions in Vibrio cholerae.     

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.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Mutagenic effect of o-methylhydroxylamine on the prophage and extracellular phage lambda

Induction of c-mutations in extracellular bacteriophage and prophage lambda cI857 ind-treated with 1 M O-methylhydroxylamine (OMHA) at 32 degrees and pH 5.6 has been studied. The frequency of c-mutations increases proportionally to the time of treatment of extracellular phage and does not depend on cellular recA+ or polA+ functions and on induction of SOS-repair system caused by UV-irradiation of host cells. Prophage is inactivated and mutagenized approximately 10-fold faster than extracellular phage immediately after treatment of lysogenic cells during prophage induction. Thus, prophage survival does not depend on repair functions of the host cells, and the frequency of c-mutations in recA and, especially, in polA lysogens is significantly lower, than in the wild-type cells.Delayed thermoinduction (90 min) of prophage causes significant enhancement of survival and decreases the frequency of c-mutations in all strains studied. Preliminary treatment of non-lysogens with OMHA does not increase the frequency of c-mutations in undamaged phage or in phage treated with OMHA in vitro.     

RecE-dependent lysogenic induction in the absence of repressor in Bacillus subtilis non-complementing diploids

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of phi 105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent phi 105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.     

An adaptive response of Vibrio cholerae strain OGAWA 154 to furazolidone

Since furazolidone is an antimicrobial drug, any possibility of its evoking an adaptive response appears to be very important. This response was studied in Vibrio cholerae cells as a model system. In order to determine this response, a dose-response relation of these cells to furazolidone and the kinetics of inactivation of the drug were studied. The study of the adaptive response of these cells to furazolidone reveals that cells treated with a low concentration of furazolidone for a particular period were 100% more resistant to the lethal effects of a subsequent challenging dose than control cultures. Variation of the challenging dose level showed better survival of adapted cells than control cells. A time-dependent response study reveals a maximum response at 15-30 min, and a gradual fall thereafter.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Preferred secondary attachment site of prophage phi80 on Escherichia coli K-12 chromosome]

The integration frequency of phage att80 immlambdac1857 into the chromosome of a mutant strain H47 Escherichia coli K-12 deleted for the normal prophage insertion site is found to be about 20-fold decreased as compared with its integration into the wild type strain. The most of the resulting lysogens contain the prophage at the secondary attachment site of the mutant bacterial chromosome which is preferentially utilized for prophage insertion. This attachment site (att80-II) is located close to his-genes on the chromosome of H47 strain. Prophage curing procedure of such abnormal lysogens results in the appearance of rare auxotrophic heat-resistant survivors with the His- phenotype. In some cases the prophage insertion can induce an inversion of a neighbouring genetic region. Such lysogens contain the purC gene near prophage located at the att80-II site, and after curing they segregate the heat-resistant His- and Pur- colonies.     

Influence of Transformability on the Formation of Superinfection Double Lysogens in Haemophilus influenzae

Superinfection of growing (nontransformable) cells of defectively lysogenic strains of Haemophilus influenzae with wild-type or with mutant phage HP1 resulted in a number of double lysogens and a small number of monolysogens with altered prophage. The double lysogens were identified by analysis of their monolysogenic segregants and by examining their deoxyribonucleic acid in certain test crosses. The results indicate that the majority had been formed by insertion of the infecting phage genome within the resident prophage. Superinfection of transformable bacteria gave rise to cells with altered prophages (presumably transformants) and to double lysogens which had gained or lost wild-type prophage loci.     

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.     

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.     

Comparison of the biopotency of corticosterone and dexamethasone acetate in glucocorticoid receptor down regulation in rat liver

The effects of long treatment with dexamethasone 21-acetate and corticosterone on the glucocorticoid receptor in rat liver cytosol were compared. Dexamethasone acetate (5 micrograms/ml or 10 micrograms/ml water) or corticosterone (100 micrograms/ml water) was given to adrenalectomized animals as drinking solution for 6 days, and glucocorticoid receptor concentration was determined at 0, 12, 24, 48 and 72 h after steroid withdrawal. Dexamethasone acetate caused a dose dependent depletion of cytosol receptor. There was no measurable binding at time 0; the values of Bmax for the glucocorticoid receptor with decreased at 12, 24 and 48 h after the steroid withdrawal. Increased dissociation constant (Kd) were calculated for 12 and 24 h samples. The effect of corticosterone on receptor depletion was less pronounced. Bmax for the receptor was decreased at 0, 12, 24 h after steroid withdrawal with no change in Kd. The extent of steroids-induced receptor depletion showed good correlation with the induction of tyrosine aminotransferase (TAT), however, maximum TAT activity measured immediately after withdrawal of dexamethasone acetate was lower than that found after a single injection of dexamethasone acetate. We conclude that both steroids cause down regulation of the glucocorticoid receptor in rat liver cytosol, with both the extent and the duration of depletion being dependent on the biopotency of the glucocorticoid.     

A simple method for making new transducing lines of coliphage lambda.

A variety of novel transducing lines of phage lambda can be obtained by induction of a mixed culture of abnormal lysogens. Such a culture is simply made by mass lysogenization of a host lacking the normal prophage attachment site.     

Prophage induction and inactivation by UV light.

Analysis of the induction curves for UV light-irradiated Haemophilus influenzae lysogens and the distribution of pyrimidine dimers in a repair-deficient lysogen suggests that one dimer per prophage-size segment of the host bacterial chromosome is necessary as a preinduction event. The close correlations obtained prompted a renewed consideration of the possibility that direct prophage induction occurs when one dimer is stabilized within the prophage genome. The host excision-repair system apparently functions to reduce the probability of "stabilizing" within the prophage those dimers that are necessary for induction and inactivation. The presence of the inducible defective prophage in strain Rd depresses the inducibility of prophage HP1c1.     

Reaction of the cell center to exposure to the calcium ionophore A-23187

Calcium ionophore A23187, taken at a concentration of 0.1 microgram/ml, quickly uncouples mitochondria in PE culture cells in medium 199. The cell ultrastructure undergoes reversible changes (especially that of mitochondria): maximum changes occur 2 hours after the start of the treatment; in 8 hours they become less pronounced. The adaptation of cells does not involve the ionophore inactivation in the medium. 10 micrograms/ml of A23187 induces gradual but irreversible alterations. Microtubules in PE cells are not destroyed when incubated in medium 199 containing 10 micrograms/ml of A23187 and 11 mM Ca2+. The addition of 10 micrograms/ml ionophore to the normal 199 medium (1.26 mM Ca2+) results in the formation of electron dense bodies in the cell center 30 minutes after the start of incubation. These bodies disappear in the course of a subsequent incubation. The number of cells with primary cilia decreases. The percentage of centrioles located perpendicularly to the substrate increases 30 minutes following treatment with 0.1 microgram/ml A23187 in medium 199. 2 hours after the start of treatment with 0.1 microgram/ml ionophore no such changes are detected; an electron dense halo appears around the centriolar cylinders. 8 hours after the start of treatment the structure of the cell center does not differ from the normal one.     

Effect of transient lambda prophage induction on ultraviolet light resistance and recombination in Escherichia coli.

Transient induction of lambda prophage increases the ultraviolet light resistance of most exponentially growing Escherichia coli lysogens. Resistance is increased in wild-type, recB, recB recC, recB recC recF, and recB recC recL hosts. No enhancement in recA lysogens was found, nor was there enhancement in stationary cultures. Enhancement was dependent upon the lambdared recombination system. Transient induction also increases the genetic recombination rate in recB lysogens as measured in Hfr X F- matings.     

Mutagenic action of nitrous acid on prophage lambda

The lethal and mutagenic effects of nitrous acid (0,1 M NaNO2 in 0,1 M acetate buffer, pH 4.6) on prophage lambda cI857 ind- were studied in the wild-type cells of Escherichia coli and in 9 repair-deficient mutants: uvrA6, uvrA6 umuC36, uvrD3, uvrE502, polA1, recA13, lexA102, recF143 and xthA9. After treatment with HNO2, the prophage was heat-induced either immediately or after 90 min incubation in broth at 32 degrees C. The prophage survival after delayed induction was considerably higher than after immediate induction. The lethal action of HNO2 was highly expressed in uvrA- and uvrE- lysogens after delayed induction. The frequency of temperature-independent c mutants forming clear plaques at 32 degrees C reached 4% in the wild-type host after immediate induction, this value being 10-15% in uvrA, uvrA umuC, uvrD, uvrE, polA and xthA mutants, 0,8% in recF- lysogen and only 0,2-0,3% in recA and lexA mutants. Under these conditions, about 90% of c mutants are generated by recA+, lexA+-dependent repair mechanism (most probably, due to W-mutagenesis). After delayed induction, mutation frequency in the wild-type host declines considerably (down to 0,1%). Analogous phenomenon of mutation frequency decline was registered in uvrA, xthA, recF, polA, uvrE and uvrD lysogens. Under conditions of delayed induction, the frequency of HNO2-induced c mutations only slightly depends on the recA+ and lexA+ gene products and mutations are, apparently, fixed by replication.