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PubMed1976

Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.

Coliphages↗
PubMed1976

Genetic studies of coliphage P1. III. Extended genetic map.

An extensive genetic map of coliphage P1 has been constructed for 113 amber mutants, using primarily a modification of the conventional complementation spot test. These spot tests failed to classify the mutants into cistrons, but when they were quantitated they permitted assignment of the mutants into 10 linkage clusters. Furthermore, a linear order could be deduced for most of the mutants within each cluster. This strongly suggested that recombination was the predominant event generating plaques and that, for the practical purpose of rapid genetic mapping, such spot tests could be considered as a series of two-factor crosses. Six of the 10 linkage clusters correlated with the P1 genetic map established by Scott (1968). The locations of the remaining four clusters were determined by three-factor crosses and by prophage deletion mapping. The nonrandom occurrence of termini for 14 deletion prophages, which we established previously (Walker and Walker, 1975), and the coincidence of these termini with five out of ten regions demarcating the linkage clusters are discussed. Complementation tests in liquid frequently gave ambiguous results. Therefore, cistron designations were not assigned.

Chromosome Mapping↗
PubMed1977

Temperate Bacillus subtilis bacteriophage phi 3T: chromosomal attachment site and comparison with temperate bacteriophages phi 105 and SPO2.

The temperate Bacillus subtilis bacteriophage phi 3T contains within its genome a locus, designated thyP3, that encodes for a protein with thymidylate synthetase activity. Bacteriophage phi 3T is different from the two previously characterized temperate phages, phi 105 and SPO2, in: heteroimmunity, response to bacteriophage antisera, endonuclease digestion pattern, induction in the presence of 6-(p-hydroxyphenylazo)-uracil, and effect on the lytic cycle of bacteriophage phi 1. The mean burst size of phi 3T is 56. The dose response curve with bacteriophage phi 3T DNA is linear for transfection and transformation to the Thy+ phenotype. The inserted prophage has been mapped by PBS1 transduction; it is between chromosomal markers ilvA8 and gltA in the terminus of the chromosome. Thus thyP3 maps at a site separate from, but between, the bacterial markers thyA and thyB when thyP3 is in the prophage state.

Bacillus subtilis↗
PubMed1977

Reproductive fitness of P1, P2, and Mu lysogens of Escherichia coli.

P1, P2, and Mu lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats. Thus, prophage-containing stains of E. coli are reproductively more fit than the corresponding nonlysogens. If mixed populations are grown by serial dilution under conditions in which growth is not limited, both the lysogen and nonlysogen manifest identical growth rates. The increased fitness of the lysogens in glucose-limited chemostats correlates with a higher metabolic activity of the lysogen as compared with the nonlysogen during glucose exhaustion. We propose that P1, P2, Mu, and lambda prophage all confer an evolutionarily significant reproductive growth advantage to E. coli lysogenic strains.

Aerobiosis↗
PubMed1977

Transduction of bacteriophage Mu by bacteriophage T1.

Phage T1 transduces phage Mu PFU from Mu-lysogenic donor cells to sensitive recipient cells. The efficiency of transduction depends on the chromosomal location of the Mu prophage. T1, therefore, appears to package different regions of the bacterial chromosome with different efficiencies. Although T1 transduces bacterial markers with different efficiencies, there is no direct correlation between the efficiency of transduction of a bacterial marker and the efficiency of transduction of Mu PFU from donor cells with the Mu prophage located in that marker.

Coliphages↗
PubMed1977

Inhibition of lytic induction in lysogenic cyanophyces.

When the lysogenic strain SPIcts1 of the blue-green alga Plectonema boryanum carrying a temperature-sensitive mutation in the LPP2 prophage was heated at a nonpermissive temperature in the light, a lytic cycle occurred, with production of infectious viral particles. Inhibitors of transcription, translation, and photosynthetic functions interfered with this process and produced different effects when administered at different phases of the viral cycle. The presence of the inhibitors during the temperature shift did not allow a successful induction to take place; lysogens submitted to such a process produced a normal virus yield, however, when the drugs were removed and the temperature was shifted again. Incubation with the inhibitors during the early postinduction period reduced the virus yield; at later times, however, the inhibitory action rapidly declined. When cells were induced in the presence of chloramphenicol, incubated with actinomycin, and then grown in the dark, at either permissive or nonpermissive temperatures, virus multiplication was equally inhibited. These data indicate that: (i) provirus induction in lysogenic cyanophyces relies on the synthesis of early viral proteins; (ii) induction of mRNA is unstable and becomes rapidly inactivated when its translation is prevented; and (iii) inhibition of photosynthesis prevents the induction message from being expressed. It is suggested that the SPIcts1 prophage codes for a mutated repressor, which is reversibly inactivated at a nonpermissive temperature, and that the repressor must be inactivated at the same time that the message coded for by very early genes is translated, for a successful induction of the lytic cycle.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1977

Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae.

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.

Bacteriophages↗
PubMed1978

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: identification of different types of specialized transducing particles.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanism. Phage lysates produced by induction of lysogenic strains contain very high frequencies of supQ newD- and proA,B-specialized transducing particles (10(-2)/PFU and 10(-3)/PFU, respectively), most of which are produced by independent aberrant excision events of various types. In a model, 12 different modes of transduction mechanisms were characterized by: (i) the structure of the specialized transducing genomes after injection into a new host cell, i.e., linear or circular, and (ii) the requirements for the transduction process, i.e., host recombination functions, phage integration functions, or presence of a prophage. By using different recipient strains and phage helper strains, it was possible to show that most specialized transducing particles (ca. 99%) contain linear genomes that cannot circularize upon injection into a new host cell and that require the presence of an integrated prophage as a site for a recombinational event to give rise to a transductant. Only 0.1% of all specialized transducing particles were shown to transduce by integration, suggesting that transducing genomes containing terminally redundant ends represent only a minor fraction of all transducing particles that are produced. However, it should be pointed out that the frequency (approximately 10(-5)/PFU) of these specialized transducing genomes that can circularize upon injection into a new host cell is as high as or even higher than the frequency of specialized transducing particles of phage lambda. The remaining approximately 1% of all specialized transducing particles can transduce by any one of the other mechanisms described.

Adsorption↗
PubMed1978

Close association between shape alteration and loss of immunity to superinfection in a wild-type Klebsiella pneumoniae stable lysogen which can be both immune and nonimmune to superinfection.

Klebsiella pneumoniae MirM7 is a wild-type strain which grows as cocci at pH 7 and above and as rods at pH 6.5 and below. Cultures of this strain and an auxotrophic derivative, MirM7b, have been found to undergo spontaneous lysis after purification from possible contaminating viruses. Lysates always contained two phages, FR2 and AP3, most often at high titers. FR2 and AP3 plated with the same efficiency on both MirM7b and K59 (another K. pneumoniae strain sensitive to FR2 and AP3) and lysogenized 45 and 54% of the K59-infected cells, respectively. These findings raise the possibility that MirM7b is lysogenic for FR2 and AP3, although nonimmune to their superinfection. The fact that mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine can induce phages FR2 and AP3 from MirM7b confirmed this possibility. When MirM7b was infected with FR2 several strains immune to FR2 and AP3, which were all rod shaped, were obtained. Furthermore, 19 derivatives, rod shaped at all pH's have been isolated from MirM7b. They were all immune to both FR2 and AP3. From mating experiments between the MirM7b donor derivative, strain M720, and either K59 or MirCV5, a rod-shaped MirM7b derivative cured from the prophages, cysteine recombinants were obtained which were most often (80%) immune to FR2 and AP3. Nonimmune and still lysogenic recombinants were obtained by mating M720 with a rod-shaped immune MirM7b derivative; the majority of the non-immune strains maintained the rod shape. Five coccus-shaped recombinants were also isolated; they were nonimmune to superinfection. Several physiological properties of strain MirM7b and the other nonimmune coccal recombinants have been studied in comparison with those of the rod-shaped immune derivatives. All of the coccal strains have shown several alterations with respect to the rods. The role of possible derepressed prophage genes in the various physiological alterations of MirM7 is discussed, and the analogies between this system and those of vertebrate cells transformed by proviruses are stressed.

Bacteriophages↗
PubMed1981

The transient inability of the conjugating female cell to host 186 infection explains the absence of zygotic induction for 186.

In an Hfr(186) X F- cross, the 186 prophage on the incoming male chromosome is not induced, despite the fact that prophage 186 can be induced by other means (W. H. Woods and J.B. Egan, J Virol. 14:1349-1356, 1974). We show here that the conjugating female is temporarily inhibitory to infection by 186, and this delay, we postulate, enables cI repression to be reestablished before the female cell recovers its 186 sensitivity.

Chromosomes, Bacterial↗
PubMed1970

Heat-sensitive early function in induced lambda Nsus lysogens.

Mutations in gene N of lambda prevent killing of the host bacterium after infection. However, derepression of Nsus prophages in nonpermissive (pm(-)) bacteria results in death of the lysogens. When prophages in pm(-)(lambdaCItsA-Nsus) lysogens are derepressed by raising the temperature to 45 C, the cells remain viable as long as they are at 45 C. However, they cannot form colonies at 33 C unless they have been superinfected, at the high temperature, by lambdaCI(+)-Nsus phage which produces repressor at 45 C. A large fraction of these "rescued," heat-inducible lysogens are lysogenized by the superinfecting phage, but lysogenization is not required for rescue. In pm(-)(lambdaCItsA-Nsus) lysogens growing at 45 C, the rate of deoxyribonucleic acid (DNA) synthesis shows a characteristic increase after the temperature is lowered. This increased DNA synthesis, which is correlated with loss of rescue potential, does not occur as long as the cultures are maintained at 45 C.

Coliphages↗
PubMed1970

Low-frequency rescue of a genetic marker in deoxyribonucleic acid from Bacillus bacteriophage phi 105 by superinfecting bacteriophage.

Markers in gene L, which maps at the right end of the vegetative and prophage maps, are rescued at a strongly reduced frequency from mature phi105 deoxyribonucleic acid (DNA) by superinfecting phage but at high frequency from vegetative and prophage DNA. It is suggested that the ends of mature DNA are degraded when DNA is taken up by competent cells.

Bacillus↗
PubMed1971

Transfection of Escherichia coli spheroplasts. I. General facilitation of double-stranded deoxyribonucleic acid infectivity by protamine sulfate.

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.

Centrifugation, Density Gradient↗
PubMed1971

Growth of bacteriophage phi 105 and its deoxyribonucleic acid in radiation-sensitive mutants of Bacillus subtilis.

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.

Bacillus subtilis↗
PubMed2026

Intra-strain genomic microevolution and phage resistance in Pseudomonas aeruginosa PAO1 laboratory isolates.

Pseudomonas aeruginosa is a major opportunistic pathogen, and its laboratory reference strain, PAO1, is widely used in microbiological and genetic studies. However, PAO1 often exhibits phenotypic variability that can affect experimental reproducibility. Our PAO1 stock, obtained from a public biobank, is resistant to PP7, a pilus-dependent single-stranded RNA phage known to infect PAO1. This suggests the presence of genetic variants in the stock. To check this possibility, we isolated six phenotypically distinct variants (GU1-GU6) and performed genomic and phenotypic analyses. Notable differences were observed among the isolates in terms of motility, pyocyanin production, and susceptibility to PP7. Whole-genome sequencing revealed that four of the six variants harbored mutations in pilus-associated genes. Among these, GU3 carried a mutation in pilT, which encodes a motor protein essential for type IV pilus retraction, and the loss of retraction led to the PP7 resistance. GU2, GU4, and GU6 shared a nonsense mutation in pilJ, a gene involved in chemotaxis and pilus extension, resulting in reduced twitching motility and lower PP7 infection efficiency. Additionally, we found that a mutation in lasR, a master regulator of quorum sensing, promoted the replication of prophage Pf6, which was integrated into the PAO1 genome. Pf6 replication interferes with PP7 infection, providing an alternative mechanism of resistance. These findings offer new insights into the complexity of phage-host interactions and emphasize the importance of routine verification and careful handling of P. aeruginosa sublines used in bacteriological and phage research.IMPORTANCEPhenotypic and genotypic variability in Pseudomonas aeruginosa PAO1 has been widely reported, raising concerns regarding the reproducibility of laboratory studies that rely on this reference strain. In this study, we isolated six PAO1 variants from a single laboratory stock and demonstrated that they differed markedly in motility, pyocyanin production, and susceptibility to the ssRNA phage PP7. Whole-genome sequencing has revealed that even a single mutation in a pilus-associated gene can profoundly affect bacterial motility and phage susceptibility. Furthermore, we showed that a mutation in lasR, a key regulator of the quorum-sensing system, triggered replication of the Pf6 prophage, which in turn hindered PP7 infection. These findings underscore the dynamic nature of laboratory strains and highlight the need for caution when interpreting results from phage-host interaction studies using reference strains. Our results provide a new understanding of how subtle genetic changes in model strains influence experimental outcomes in microbiology.

Pseudomonas aeruginosa↗
PubMed1981

Some properties of a defective bacteriophage of Bacillus brevis.

Bacillus brevis strains contains defective prophage. In strain ATCC 10068 this prophage is responsible for the production of particles resembling phage tails and the induction of events leading to cell lysis. There is a slow rate of spontaneous production of particles, which is greatly increased by treating growing cells with mitomycin C, ultraviolet light, N-methyl-N-nitroso-N'-nitroguanidine, and acridine orange or by changing the growth medium. There is no conversion of host DNA to a unique, phage-size DNA entity associated with induction. It was not possible to detect strains that did not produce particles on treatment with mitomycin C. The particles themselves had no recognised biological activity. The molecular weight of the major sheath protein was 49 000 and at least four other protein components were detected in partially purified preparations. DNA and RNA were not associated with the particles but a small amount of carbohydrate was detected.

Bacillus↗
PubMed2004

Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12.

BACKGROUND: An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage lambda Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating lambda vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants. RESULTS: A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case. CONCLUSIONS: The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.

Bacteriophage lambda↗
PubMedDate not supplied

Inducibility of lambda phage development in Escherichia coli mutants thermosensitive for DNA replication.

In a thermosensitive dnaZ mutant lysogenic for lambda, induction of the prophage development was provoked at the restrictive temperature. In dnaD strain, spontaneous release of lambda proceeded even at 43 degrees C, but distinct induction of the prophage development was not caused by a heat treatment. Similarly, shift up of growth temperature did not lead to induction of lambda in lysogens carrying thermosensitive mutation in dnaH function or DNA polymerase I. In dnaH mutant, multiplication of lambda-virulent phage proceeded normally at 43 degrees C.

Coliphages↗