Clear-plaque Mutants Research Articles

Lysogenization by satellite phage P4

Satellite phage P4 attaches to the E. coli K-12 chromosome at a preferred site near 96 min. The attachment site on the P4 genome is 31.4–36.5% from the left cohesive end, which has been redefined as the end with the same base sequence as the P2 helper phage left end. The P4 int gene is in the region from 27.5 to 31.4%. Immunity-sensitive clear plaque mutants define two P4 genes. One is likely to code for repressor and maps very near the P4 gene which is needed to derepress a helper prophage. The other is probably not involved in lysogenization and maps near the left end of the P4 genome, among nonessential genes.

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta.

The restriction fragment patterns of two mutants forms of the temperate Bacillus subtilis bacteriophage SP beta have been examined. The DNA of a heat-inducible mutant, SP beta c2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SP beta c+ DNA. The DNA of a clear-plaque mutant, SP beta c1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SP beta c1 nor SP beta c2 DNA is cleaved by the endonuclease HaeIII.

RESTRICTION ENDONUCLEASE ANALYSIS OF DNA FROM THE STREPTOMYCES PHAGES SH3, SH5, SH10 AND SH13

Abstract Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only Eco RI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by Eco RI, Kpn I and Bgl II, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clearplaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two Kpn I sites.

Restriction endonuclease analysis of DNA from the Streptomyces phages SH3, SH5, SH10 and SH13

Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only EcoRI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by EcoRI, KpnI and BglII, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clearplaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two KpnI sites.

Detection of mutagen-induced lesions in isolated DNA by marker rescue of Bacillus subtilis phage φ105

A new mutagenesis assay is described which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative from DNA of the temperate Bacillus subtilis phage φ105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. There is a cluster of such genes tightly linked to the φ105 genetic marker Jsus11 which restricts the host range of the phage to cells capable of suppressing sus mutations. In the actual assay chemically treated DNA, from wild-type J + phage, is added to competent cells which are infected with φ105 Jsus11. Wild-type phage, capable of producing plaques on cells which are nonpermissive for φ105 Jsus11, are produced by recombination between the added chemically-treated DNA and infecting φ105 Jsus11 DNA. If the added DNA also carried mutagenic lesions in any of the genes controlling lysogeny, clear plaque mutants are produced which are readily distinguishable from the turbid plaquing wild-type phage. This report demonstrates the capacity of this marker rescue-based assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl- N′-nitro- N-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl- N-acetoxy-2-amino-fluorene (AAAF). The effect of using a host cell, defective for excision repair, on the sensitivity with which the assay detected the mutagenic activities of CAA, PO and AAAF also was examined. The new mutagenesis assay offers 2 advantages over several other previously described transformation-based assays: (1) in contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution-type mutagens and (2) the mutants generated can be detected at high plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.

A clear-plaque mutation of bacteriophage M13 affects the regulation of viral DNA synthesis

A clear-plaque mutation (c2) of bacteriophage M13 has been shown to affect the regulation of viral DNA synthesis. This mutation increases the amount of the duplex replicative form DNA per cell while decreasing the synthesis of viral single strands. The relative synthesis of the M13 gene 5 protein is approximately half that observed in wild-type infections, suggesting that the effect of the c2 mutation on the regulation of viral DNA synthesis is a result of reduced expression of gene 5.

Survival and mutagenesis in UV‐irradiated phage: Multi‐hit kinetics of mutation induction and lack of indirect induction by infection with UV‐irradiated phage of error‐prone repair

AbstractThe paper is concerned with the question of whether Weigle‐reactivation (WR) and Weigle‐mutagenesis (WM) can be indirectly induced by infection with UV‐irradiated phage. Experiments neither with phage λ of Escherichia coli nor with phage χ of Serratia marcescens show such induction‐In this respects phage DNA differs from F′‐DNA or Hfr‐DNA; possible explanations are discussed.In both systems clear plaque mutations can also be induced by UV without irradiation of the host cells; they appear, in unirradiated and irradiated host cells, with an increase in frequency which is greater than proportional to the UV dose. It is concluded that mutation induction of phage in the unirradiated host cells is due to a low level constitutive mutagenic repair; this could either be due to “spontaneous” induction of the mutagenic SOS‐function or it could be a mechanism different from this one. Host irradiation would give rise to additional activity by the induced SOS‐function leading to WR and WM.It is further concluded that deviation of the induction kinetics from a linear dose‐dependence is not due to the necessary induction of SOS‐functions.

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process.

Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA. Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.

Characterization and persistence of actinophage RP2 isolated from Streptomyces rimosus ATCC 10970.

While searching for true lysogens among oxytetracycline-producing Streptomyces rimosus strains, free phage particles were detected and isolated from a liquid culture of S. rimosus ATCC 10970 (R7). The actinophage, designated RP2, appears to be a typical temperate DNA phage producing turbid plaques on the sensitive strain S. rimosus R6. Electron microscopic examination of RP2 lysates showed that it belongs to group B of Bradley's morphological classification. The rate of RP2 adsoprption at 28 degrees C appeared to be low. The length of the latent period was about 6 h and the average burst size about 120 phage particles. The lysogenic nature of the host-virus system described was established on the basis of the following characteristics: spontaneous lysis frequency of 2 X 10(-6) per cell, resistance to curing with phage-specific antiserum, spontaneous curing frequency of less than 0.05% and immunity to superinfection with the homologous phage. Clear-plaque mutants of RP2, which failed to lysogenize sensitive cultures, arose at a frequency of 10(-5).

The prophage of SP beta c2dcitK1, A defective specialized transducing phage of Bacillus subtilis.

The defective specialized transducing phage SP beta c2dcitK1 carries two known bacterial genes, kauA and citK, as well as SP beta hage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SP beta c2dcitK1 transduces SP beta-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSP beta-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and of the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SP beta c2 or by the clear-plaque mutant SP beta c1, the resulting double lysogen can be heat induced to release high-frequency-of-transduction (HFT) lysates for kauA and citK.

Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.

ANALYSIS OF THE ROLE OF RECOMBINATION AND REPAIR IN MUTAGENESIS OF ESCHERICHIA COLI BY UV IRRADIATION

Multiple mutant strains have been tested or their mimicry of the UV-mutagenesis deficiency of a recA single mutant. Revertants to histidine prototrophy and clear plaque mutants of lambda were scored to determine capacity for UV-mutagenesis. Nearly normal capacity was shown by a uvr+ recB- recF- strain, which shows almost no recA-dependent recombination, by uvr- recB+ recF- strains, which show almost no recA-dependent repair and by a uvrA- recB- recF- strain, which shows neither recA-dependent recombination nor repair. Since the uvr mutants can be assumed to show additionally no excision repair, these results may mean that UV-mutagenesis occurs during processes other than recombination and repair. Alternative hypotheses are discussed. The slight difference in mutagenic capacity was traced to the recF single mutation, which blocks the production of unmixed bursts of clear-plaque lambda mutants. Since this accounts for only about 10% of the mutations leading to clear-plaque mutants, it is suggested that there is more than one UV-mutagenic process.

Clear plaque mutants of phage P7

Clear plaque mutants of the P1-related phage P7 were isolated and classified by complementation tests with one another and with the P1 clear plaque mutants. In addition to several types of virulent mutants, a total of eight complementation groups of clear plaque mutants has been identified in these phages. A class of nonvirulent clear mutants unable to complement either P7c1 or P7c4 was also found. Mutants in this class have no genetic defect in the c4 region and are probably single mutants. Like c1 ts lysogens of P1, P7c 1 ts lysogens are thermally inducible, which supports the idea that gene c1 of both phages controls a repressor. P7c4 mutants cannot be complemented by any P1 clear mutants and P1c4 mutants cannot be complemented by any P7 clear mutants. This complementation behavior is expected if gene c4 codes for the immunity specific repressor in each of these heteroimmune phages. Unlike P1c4ts lysogens, P7c4ts lysogens are thermally induced to produce phage. This difference is discussed, but has not yet been explained.

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.

Temperate coliphage HK022. Clear plaque mutants and preliminary vegetative map.

Wild type phage HK022 was mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine to induce clear plaque mutants. A total of 225 clear plaque mutants were isolated and 198 of these were assignable to one or the other of two complementation groups of the corresponding cistrons which have been designated as cI and cII, respectively. Approximately 25% of the c mutants were found to be temperature-sensitive (cts); producing turbid plaques at 32 C and clear plaques at 38 C and above. From complementation tests involving cI and cII mutants, bacteria lysogenic for cII prophage were frequently obtained. Double lysogens harboring a CI and a cII prophage were infrequently found and single lysogens harboring only a cI prophage have not been recovered. Bacterial lysogens harboring a prophage carrying a cts mutation in the cI cistron were readily obtainable. However, such lysogens show a lethal phenotype at 40 C and above, although they appear to be fully viable at 32 C. It is shown that by incubation of lysogens harboring a cts mutant of the cI cistron at 42 C, it is possible to isolate cryptic lysogens which are non-immune but harbor at least one of the phage sus+ alleles. Genetic data involving cI, cII, and two complementing sus mutants of essential genes are presented. From these data the following vegetative map is deduced: sus4--cII-cI-sus3.

Transport in bacteriophage P22-infected Salmonella typhimurium.

There was rapid efflux of L-leucine, L-phenylalanine, and alpha-methyl-D-glucoside after infection of Salmonella typhimurium with the clear plaque mutant C1 of phage P22. The efflux was similar to that observed with cyanide or arsenate treatment except that there was partial recovery in the case of phage infection and almost complete recovery under the condition of lysogeny. There was no efflux after infection with the temperature-sensitive mutant ts16C1 at nonpermissive temperature. Superinfection of superinfection exclusion negative lysogen (sie A minus sie B minus) with C1 led to efflux, whereas the efflux was much less on superinfection of sie A+ Sie B+ lysogen. These results indicate that an effective injection process is enough to cause depression in the cellular transport processes.

Events following prophage Mu induction.

Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.

Induction and mutagenesis of prophage λ in Escherichiacoli K12 by metabolites of aflatoxin B 1

Like most carcinogens, aflatoxin B 1 must be activated by mammalian microsomal enzymes to give rise to compounds active on bacteria. These compounds act as inducers of E. coli K12 (λ)_at a high efficiency, whereas unmodified aflatoxin B 1 has no effect. Moreover, metabolites of aflatoxin B 1 have a mutagenic action on phage λ, as shown by the appearance of clear plaque mutants. We propose the hypothesis that the same derivatives is responsible for carcinogenesis of liver cells by aflatoxin B 1. Therefore, our system provides a simple way of measuring in vitro, in the same assay, the mutagenic and inducing activities of compounds to which the cells are permeable, thereby detecting potentially carcinogenic agents.

Antigenic relationships among Newcastle disease virus mutants obtained from laboratory strains and from recent California isolates.

The antigenic relationship between pairs of plaque mutants of Newcastle disease virus (NDV) derived from laboratory strains and from isolates from the 1971-72 California epizootic were examined by kinetic neutralization test. Comparing four sets of mutants from laboratory strains by both kinetic neutralization and hemagglutination inhibition tests, a similarity was found in the antigenic relationship expressed as an r value with both tests. However, kinetic neutralization was the more precise as well as sensitive assay. Antigenic diversity was greatest between pairs of mutants from different strains, but distinctions could also be made between mutants from the same strains such as Herts-L and Herts-S with an r value of 36%. Examination of mutants from the California epizootic isolated from separate locations and at different times showed antigenic divergence which was greatest between two red-plaque mutants with an r value of 39%. Antigenic distinctions were found between a red- and clear-plaque mutant obtained from isolates taken from brain and tracheal swabs of one infected chicken. In addition to antigenic divergence found between pairs of some mutants, two of the clear-plaque mutants reacted more avidly with antibody than did the corresponding red-plaque partner. Thus, both differences in antigenicity and avidity can be found among these NDV mutants. The antigenic variation found among these mutants is similar to that found within a serotype. This would imply that at the present NDV is a single serological type.

Abortive infection of lysogenic Bacillus subtilis 168(SPO2) by bacteriophage phi 1.

Virulent bacteriophage phi1 was not able to productively infect strains of Bacillus subtilis which were lysogenic for the temperate bacteriophage SPO2, although it adsorbed to, penetrated, and killed these bacteria. Studies of phage and host nucleic acid production in the nonpermissive host demonstrated that normal phi1 transcription was initiated early in the latent period, but this was followed by a general failure of host and phage nucleic acid synthesis about 10 to 15 min after infection. Mixed infections of phi1 and SPO2c(1), a clear-plaque mutant of SPO2, indicated that a similar inhibition of phi1 development occurred when this phage infected nonlysogenic B. subtilis cells committed to the SPO2c(1) lytic cycle. It is proposed that the SPO2- and SPO2c(1)-mediated interference did not act directly on the phi1 genome, but rather these phages altered the host physiology in such a manner that some normal step in phi1 development triggered a collapse of vital metabolic activities.