Phage Synthesis Research Articles

Role of dna genes of Escherichia coli in M13 phage replication

Escherichia coli mutants, all lacking DNA polymerase I and having single temperature-sensitive mutations in the five known dna genes, have been tested for their ability to support M13 phage synthesis at the restrictive temperature. The dnaA. product is required early after infection. Neither dnaA nor dnaB product is required late after infection. The products of dnaC (D), dnaE and dnaG genes are all required continuously for phage production. Unlike the effect of dnaE mutation, the effects of dnaC ( D) and dnaG mutations on phage production, as well as on host DNA synthesis, are reversed by temperature shift-down. Unlike the wild-type strain, an endonuclease I- and DNA polymerase I-deficient strain with no temperature-sensitive lesion in DNA synthesis used as the control in the present studies is abortively infected at the restrictive temperature (44 °), but behaves like the wild type when the temperature is shifted up to 44 ° early after infection at permissive temperature.

Regulation of bacteriophage T4 gene expression

The programme of bacteriophage T4 gene expression and its control by DNA synthesis and by the expression of maturation product function have been investigated in detail by the analysis of RNA, protein synthesis and the synthesis of intracellular phage. Single amber maturation-defective mutants in gene 33 are shown not to block maturation totally. Since single amber mutants can be leaky, multiple amber mutants defective in DNA synthesis and maturation product functions are used here. It is known that mutually complementary transcripts are made during T4 development. The experiments described here show that the anti-late RNA from DNA l-strand appears at two minutes after infection at 30 °C, at the same time as delayed early RNA. Anti-late RNA reaches a steady-state concentration at four minutes. It constitutes 2% of the total T4 RNA present in the 5-min RNA and it is at least as stable as the l-strand-specific phage messenger RNA. The DNA template contributing to the transcription of anti-late RNA is estimated to be at least 81% of that DNA l-strand which is complementary to the entire length of the DNA r-strand involved in the transcription of late RNA. Prereplicative r-strand transcript constitutes only 0.1% of the total prereplicative phage RNA. The increased synthesis of quasi-late RNA from the DNA l-strand starts to occur between six and ten minutes after infection. The r-strand late transcription starts between 10 and 12 minutes after infection and all the late RNA is transcribed at least once in this two-minute interval. The observation that at least 14 new protein bands (late proteins) first appear between 11 and 14 minutes is consistent with these observations on the synchronous initiation of late RNA species. Studies by other investigators using single amber DNA negative and maturation defective mutant infections show that (1) quasi-late RNA content of 20-min DO † † Abbreviations used: D0, DNA negative; MD, maturation deficient. and MD-infected cells is similar to that of 20-min am + RNA and that (2) all the DNA l-strand late RNA species are present in lower amounts in the 20-min MD RNA. Using multiple mutants in the present studies, the l-strand quasi-late RNA content in the 20-min D0 and MD RNA is found to be intermediate between that in 5 and 20-min am + RNA and the absolute requirement of maturation product function is demonstrated for the l-strand late RNA transcription. DNA synthesis is shown to be required for the initiation of l-strand late transcription and the continued synthesis of the l-strand RNA is dependent on continued replication. Although D0 and MD infections have the same phenotype as far as the synthesis of late RNA and of late proteins is concerned, the accentuation of some of the early RNA species and of at least two early proteins are shown to differ. In experiments performed with a temperature-sensitive mutant in DNA polymerase (gene 43), late transcription has been shown to begin within one minute after the temperature shift down from the non-permissive to the permissive temperature; and within two minutes of the temperature shift about two-thirds of the maximum amount of late RNA is synthesized. These results are interpreted to indicate that the initiation of late RNA synthesis begins synchronously without a lag, suggesting that the synthesis of late RNA may occur on the parental DNA strand under these conditions.

Inhibition of M13 phage synthesis by rifampicin in some rifampicin-resistant Escherichia coli mutants

In certain Escherichia coli strains that have altered RNA polymerases and are resistant to low levels of rifampicin, M13 phage synthesis is almost totally inhibited by the drug at concentrations that do not affect significantly the bacterial growth rate. Rifampicin does not prevent release of progeny phage particles from the cells, and its effect is reversible. The inhibition of phage synthesis is due to inhibition of phage mRNA synthesis by the drug.

Specific suppression of mutations in genes 46 and 47 by das, a new class of mutations in bacteriophage T4D.

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.

Heat induction of prophage phi 105 in Bacillus subtilis: replication of the bacterial and bacteriophage genomes.

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.

Replication of bacteriophage phiX174 in a temperature-sensitive deoxyribonucleic acid host cell.

Bacteriophage phiX174 is unable to replicate in Escherichia coli t3 at the restrictive temperature. However, if progeny phage synthesis is initiated at the permissive temperature, it will continue after a shift to the restrictive temperature.

Loss of the capacity of UV-irradiated Escherichia coli B/r to grow T4D

The capacity of UV-irradiated Escherichia coli B/r cells grown on glycerol to support the growth of phage T4 was determined at intervals during postirradiation incubation of the cells. During the first 30 min capacity was virtually unimpaired, but then a rapid decrease occurred, followed by a slow increase. The dose response curve for the minimum fraction of infective centers observed at the end of the decline phase followed that for the survival of B/r for doses up to 300 ergs/mm 2. At higher doses the relative sensitivity of the minimum infective centers decreased. After a dose of 520 ergs/mm 2 to B/r cells, phage T4 both adsorbed to them and injected its DNA normally, regardless of the time of postirradiation incubation. After infection, individual cells either supported no phage synthesis, or a nearly normal burst was produced. Phage growth appeared to occur only in those cells that could eventually form colonies. In those cells fated not to survive, there was essentially no synthesis of phage DNA, nor was phage-induced endonuclease activity observed. Since the UV dose used causes respiration to be turned off about an hour after irradiation, we suggest that for glycerol-grown cells the loss of capacity is associated with loss of respiratory activity. Under conditions where respiration is only slightly and transiently affected by UV, as in the case of glucose-grown cells, there results a smaller but significant loss of capacity. Capacity seems to be particularly sensitive to respiratory failure after UV, but in glucose-grown cells, where respiration is not turned off, capacity may be lost for other reasons.

Lysis defective mutants of bacteriophage lambda: Genetics and physiology of S cistron mutants

Five lysis-deficient isolates of the S type, (including sus, def, and one ts mutant), were characterized genetically and physiologically. All the isolates were found to share two distinctive phenotypic properties; susceptibility of host cells to chloroform-induced lysis, and overproduction of phage relative to wild-type or R-mutant yields. Complementation tests demonstrated that the sus and def isolates belong to the S cistron. Two- and three-factor genetic crosses showed that all five markers are closely linked, and located the S cistron between cistrons Q and R. Comparison of the kinetics of production of phage and phage components (serum-blocking material and DNA) by S and R mutants and wild-type phage, showed that overproduction by S mutants is achieved by prolongation of component synthesis and maturation. The failure of R mutants to overproduce even though the host cells do not lyse shows that expression of the S function is responsible for the cessation of phage synthesis at the end of the normal latent period.

The association of host and phage DNA with the membrane of Escherichia coli

The attachment of DNA to membrane in T4-infected cells was investigated by means of the Mg 2+-sarkosyl crystals technique. Both parental and progeny phage DNA are associated with cell membrane throughout the eclipse period and are subsequently detached during the synthesis of mature phage. The initial attachment process does not require protein synthesis. Detachment appears to be due to a “late function” since it does not occur following infection with a DO mutant (amN82) or after treatment with chloramphenicol or tetracycline added 9 min after infection. Host DNA remains attached to membrane during the initial stages of infection and neither superinfection nor the addition of chloramphenicol alters this association.

Effect of spermine on host-cell lysis and reproduction by a lactic streptococcal bacteriophage.

A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

Effect of Spermine on Host-Cell Lysis and Reproduction by a Lactic Streptococcal Bacteriophage

A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4.

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens V-1 is temperature-sensitive. At 25 C and 30 C, phage was released in a ratio of 1 plaque-forming unit per 100 bacteria; at 35 C, although bacterial growth was not inhibited, phage release was suppressed. Phage synthesis was induced by heat shock, 42 C for 30 min, ultraviolet irradiation, and mitomycin C. Induction by ultraviolet light was unusual-an immediate rise in phage titer followed irradiation. A large increase occurred after a 90-min latent period. The lysogenic strain was cured of the phage by incubation at 37 C, and the cured strain produced plant tumors.

Replication of coliphage M-13. II. Intracellular deoxyribonucleic acid forms associated with M-13 infection of mitomycin C-treated cells.

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F(+), hcr(-), thy(-)) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with (3)H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of phiX174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.

Intracellular development of bacteriophageφR: I. Host modification of the replicative process

During the intracellular development of φR in Escherichia coli, replicative intermediates are synthesized with properties similar to the RF † † Abbreviations used: φX, φX174; RF, replicative form DNA; φX-RF, the RF prepared from cells infected with φX174; φR-RF, the RF prepared from cells infected with φR; RF(I), replicative form DNA in which both strands are covalently closed rings; RF(II), replicative form DNA in which one strand is open, the other a covalently closed ring. of φX174. When the RF is prepared from φR-infected Salmonella typhimurium, however, differences are noted which suggest that a major fraction of this DNA is fragmented: the parental RF is predominantly in the double-stranded closed ring form, but most of the progeny RF is defective in that neither strand appears to be an intact ring. The effect of these host-induced changes in the structure of the RF may be reflected in the limited synthesis of mature phage which is also characteristic of φR development in S. typhimurium.

Production de particules douees a la fois de proprietes infectieuses et transductrices par des souches d' [formula omitted] lysogenes defectives par suite d'une deletion chromosomique

Resume Some defective lysogens of Escherichia coli resulting from chromosomal deletions extending into the integrated prophage O 80 are still able to produce infectious particles but at a very low rate. In most cases, such infectious particles have acquired the ability to transduce the genes governing tryptophan synthesis and even, in some cases, the gene cys B . It is suggested that the prophage in such strains still carries the information for active phage synthesis but that, owing to the deletion of one end of the integrated prophage, circularization and detachment can only occur by means of a crossing-over incorporating a segment of the bacterial chromosome.

Über infektiöse substrukturen aus Escherichia coli bakteriophagen : VII. Formation of a biologically intact replicative form in ribonucleic acid bacteriophage (M12)-infected cells

An intact (i.e. infectious) replicative form of M12 RNA has been separated from single-stranded bacteriophage RNA by a selective adsorption method using methylated albumin-coated silicic acid as chromatographic material. At appropriate salt concentrations of the buffer used, the single-stranded phage RNA adsorbs to this material and the replicative form remains almost quantitatively in solution. Infectious replicative form, purified by this method, was found to be uniform throughout the replication of the phage as regards its adsorption properties to the chromatographic material and its sedimentation properties in a sucrose gradient. The kinetics of the formation of this biologically intact replicative form are presented. Its main increase is found between 10 and 20 minutes after infection; this is prior to the synthesis of progeny single-stranded phage RNA, which starts at 30 minutes. From this time on, the amount of replicative form is constant during the synthesis of progeny RNA and progeny phage until lysis.

Effect of 5-Fluorouracil on the Growth of Bacteriophage R17

Graham, A. F. (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and Clare Kirk. Effect of 5-fluorouracil on the growth of bacteriophage R17. J. Bacteriol. 90:928-935. 1965.-When added to Escherichia coli within 2 min after phage R17, 5-fluorouracil (FU), at a concentration of 10(-4)m, completely inhibited the synthesis of infectious ribonucleic acid (RNA) and phage. If the addition of FU was made later than 5 min after infection, infectious RNA synthesis was blocked but infectious phage was still formed; the infectious RNA made before the addition of FU continued to be incorporated into mature phage. These properties of the inhibitor were used to determine the kinetics of phage RNA synthesis and the size of the phage precursor RNA pool. At a concentration of 2.2 x 10(-5)m FU, the yield of phage was reduced to 15% of that in an uninhibited control, 28% of the phage RNA uracil was replaced with FU, and the specific infectivity of the phage was unaltered.

The synthesis of phage and host dna in the establishment of lysogeny

A method of extraction is described which prevents excessive fragmentation of bacterial DNA. The larger bacterial DNA can then be separated from the phage DNA on sucrose gradients. The relative amounts of phage and host DNA made at various times during infection of Salmonella typhimurium with phage P22 leading to lysogeny have been determined. Phage-specific DNA synthesis begins at about 4 minutes, continues to increase for 2–4 minutes, and then decreases until complete repression is attained at 16 minutes. Autonomous phage DNA synthesis remains repressed from then on. Bacterial-specific synthesis proceeds at a decreasing rate until about 16 minutes, when almost complete inhibition is reached. When DNA synthesis recovers in the infected cells, only host-specific material is made. A new species of DNA, which is made only when phage DNA is synthesized, has been detected. The possible implications of these findings in the understanding of the lysogeny interaction is discussed.

OBSERVATIONS ON THE GROUP C STREPTOCOCCAL BACTERIOPHAGE AND LYTIC ENZYME SYSTEM.

Fox, Eugene N. (La Rabida-University of Chicago Institute, Chicago, Ill.), and Masako K. Wittner. Observations on the group C streptococcal bacteriophage and lytic enzyme system. J. Bacteriol. 89:496-502. 1965.-The phage-associated lytic enzyme of group C streptococci was assayed by measuring the solubilized portion of radioactive cell walls. By this sensitive assay system, the induced synthesis of the lytic enzyme was observed intracellularly during phage infection; at least half of the total enzyme was synthesized and remained intracellular during the eclipse period, and was then released with the liberation of mature phage. Lytic enzyme could be detected in only two of eight lysogenic strains during temperate-phage production after ultraviolet induction. Virulent phage purified by density-gradient centrifugation contained lytic enzyme presumably associated with the virus per se. No hyalyronidase was detected in association with the phage, nor was this enzyme induced during phage synthesis. Variant strains of group C streptococci, no longer serologically active, were isolated as phage-resistant mutants. These strains still adsorbed the phage, but without subsequent virus reproduction, indicating that the group polysaccharide was not the primary receptor for the virus.