Decay of incorporated radioactive phosphorus during reproduction of bacteriophage T2

The multiplication of vegetative T2 bacteriophage in B/r bacteria has been followed by studying the lethal effects of decay of incorporated radiophosphorus P³² at various stages of the eclipse period. Experiment I. Non-radioactive B/r bacteria were infected with highly radioactive (i.e. P³²-unstable) T2 and infection allowed to proceed at 37°C. for various numbers of minutes before freezing the infected cells and storing them in liquid nitrogen. The longer development had been allowed to proceed at 37°C. before freezing, the slower the inactivation of the frozen infective centers by P³² decay. Samples which were frozen after incubation for 9 minutes were completely stable. Experiment II. Radioactive B/r bacteria in radioactive growth medium were infected with non-radioactive (i.e. stable) T2 and incubated for various lengths of time before being frozen and stored in liquid nitrogen, like those of Experiment I. In this case, the infective centers were stable to P³² decay as long as they were frozen before the end of the eclipse period. The T2 progeny phages issuing from the infected bacteria were P³²-unstable. Experiment III. Radioactive B/r bacteria in radioactive medium were infected with radioactive (i.e. P³²-unstable) T2 and otherwise incubated and frozen like those of the first two experiments. In this case, the same progressive stabilization, of the infective centers towards inactivation by P³² decay was observed as that found in Experiment I. The ability to yield infective progeny of infected bacteria incubated for 10 minutes at 37°C. before freezing could no longer be destroyed by P³² decay. The progeny issuing from the infected cells were as unstable as the parental phage. These results could be explained by one of three general hypotheses. As vegetative phage begins to multiply, it is possible that: (a) there is a high probability that any part of the vegetative phage already duplicated can be saved after its destruction by P³² decay through a process analogous to multiplicity reactivation or, (b) there occurs a change in state of the deoxyribonucleic acid (DNA) preliminary to or in the course of its replication that renders it refractory to destruction by P³² decay, or, finally (c) there occurs a transfer of the genetic factors from the DNA of the infecting phage to another substance not sensitive to destruction by P³² decay.     

Regulation of Bacteriophage T5 Development by ColI Factors

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb(+) cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb(+) cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

The effect of chloramphenicol on deoxyribonucleic acid synthesis and the development of resistance to ultraviolet irradiation in E. coli infected with bacteriophage T2

To elucidate the role of protein synthesis in DNA formation, E. coli R2 infected with phage T2 was studied as a model, employing chloramphenicol to inhibit protein synthesis. The following results were obtained. 1. Chloramphenicol inhibited protein synthesis but not synthesis of nucleic acids in uninfected bacteria. 2. Studies of the effect of chloramphenicol on phage maturation indicated a delay of 2 minutes between time of addition and cessation of phage growth. 3. The increase of DNA in phage-infected bacteria was completely suppressed by the addition of chloramphenicol within 2 minutes following infection. Addition at later times showed progressively less inhibitory action depending upon the time interval, and addition after the 10th or 12th minute showed no appreciable effect on DNA synthesis despite the cessation of intracellular phage formation and protein synthesis. 4. When chloramphenicol was added to infected cells the increase of resistance to UV stopped within 2 minutes, whether or not DNA synthesis continued. Thus evolution of resistance paralleled the rate of DNA synthesis achieved, but not the amount of DNA accumulated. 5. We conclude that in infected bacteria, protein synthesis is necessary to initiate DNA synthesis but is not essential for its continuation. The resistance to UV that characterizes infected cells near the midpoint of the latent period is not due to accumulation of DNA, but depends on some chloramphenicol-sensitive process (probably protein synthesis) completed at about the time the rate of DNA synthesis becomes maximal.     

Water flow through frog gastric mucosa

To elucidate the role of protein synthesis in DNA formation, E. coli R2 infected with phage T2 was studed as a model, employing chloramphenicol to inhibit protein synthesis. The following results were obtained. 1. Chloramphenicol inhibited protein synthesis but not synthesis of nucleic acids in uninfected bacteria. 2. Studies of the effect of chloramphenicol on phage maturation indicated a delay of 2 minutes between time of addition and cessation of phage growth. 3. The increase of DNA in phage-infected bacteria was completely suppressed by the addition of chloramphenicol within 2 minutes following infection. Addition at later times showed progressively less inhibitory action depending upon the time interval, and addition after the 10th or 12th minute showed no appreciable effect on DNA synthesis despite the cessation of intracellular phage formation and protein synthesis. 4. When chloramphenicol was added to infected cells the increase of resistance to UV stopped within 2 minutes, whether or not DNA synthesis continued. Thus evolution of resistance paralleled the rate of DNA synthesis achieved, but not the amount of DNA accumulated. 5. We conclude that in infected bacteria, protein synthesis is necessary to initiate DNA synthesis but is not essential for its continuation. The resistance to UV that characterizes infected cells near the midpoint of the latent period is not due to accumulation of DNA, but depends on some chloramphenicol-sensitive process (probably protein synthesis) completed at about the time the rate of DNA synthesis becomes maximal.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C III. Inhibition of TSP-1-Specific Deoxyribonucleic Acid Synthesis at 37 C and 45 C

The effect of temperature on phage-specific deoxyribonucleic acid (DNA) synthesis was studied in TSP-1-infected Bacillus subtilis. This was facilitated by selectively inhibiting host DNA synthesis with 6-(p-hydroxyphenylazo)-uracil. The results indicated that TSP-1 DNA synthesis did not continue at 37 C and was immediately shut down after transfer to this temperature. Incubation at 45 C greatly reduced TSP-1 DNA synthesis. Phage-specific DNA synthesis could resume at 53 C, however, when the infected culture was returned to 53 C after a 2-min incubation period at 37 C. The results suggest that the inhibition of phage DNA synthesis at 37 C is reversible. Since infected cultures returned to 53 C after 2 min at 37 C could not complete the replicative cycle, the irreversible inhibition of yet another intermediate step was suggested.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Organization of Gene Function in Bacillus subtilis Bacteriophage SP82G

A generalized assessment of the functions of 26 genes of SP82G, a bacteriophage of Bacillus subtilis, has been made. The production of phage-specific deoxyribonucleic acid (DNA), DNA-filled phage heads, completed phage particles, phage-specific antigen, and developmental aberrations has been examined in lysates of temperature-sensitive mutants grown under selective conditions. The genes show a tendency to occur on the genome in three groups of related function: genes involved with DNA synthesis, with tail synthesis, and with head synthesis.     

Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.     

Spackle and Immunity Functions of Bacteriophage T4

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s⁻ infections is intermediate between that of imm⁻ and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm⁻ phage are completely deficient, whereas infections with the s⁻ phage are only partially deficient compared to wild-type infections. Whereas s⁻-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm⁻ phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm⁻-infected cells are lysis inhibited, whereas s⁻-infected cells do not show this property. Superinfecting phage inject their DNA into imm⁻-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s⁻ phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.     

Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

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 ³H-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 X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

Reversible Repression of Early Enzyme Synthesis in Bacteriophage T4-infected Escherichia coli

A mutant of Escherichia coli B, defective in its accumulation of K⁺, was found to synthesize protein at a rate proportional to the level of this cation in the growth medium. When bacteriophage T4-infected cells were incubated in growth medium containing 1 mm K⁺, phage deoxyribonucleic acid (DNA) was synthesized at a rate 25% that of normal, and phage protein was synthesized at a rate of 50% of normal. Deoxycytidine pyrophosphatase, a phage-directed early enzyme, shut off at a level of 55% that of normal when infected cells were incubated in medium containing 1 mm K⁺. However, deoxycytidine pyrophosphatase synthesis resumed in these cells when they were shifted to medium containing the normal K⁺ concentration (33 mm). DNA synthesis also attained the rate characteristic of this K⁺ concentration. These results suggest that phage DNA synthesis is not sufficient to repress early protein formation and also indicate that the inhibitor of early protein formation is an early function whose synthesis is sensitive to the same repression as that of the early proteins.     

The nucleotide sequence of bacteriophage T5 glutamine transfer RNA

Uniformly ³²P-labeled phage-specific tRNA^Gln has been isolated from bacteriophage T5-infected Escherichia coli cells and its nucleotide sequence has been determined using thin-layer chromatography on cellulose to fractionate the oligonucleotides. The sequence is: pUGGGGAUUAGCUUAGCUUGGCCUAAAGCUUCGGCCUUUGAAGψCGAGAUCAUUGGTψCAAAUCCAAUAUCCCCUGCCA_OH. The main feature of this tRNA is the absence of Watson-Crick pairing between the 5′-terminal base and the fifth base from its 3′-end. The structure of tRNA was confirmed by DNA sequencing of its gene.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

The intracellular growth of bacteriophages. I. Liberation of intracellular bacteriophage T4 by premature lysis with another phage or with cyanide

A method is described for liberating and estimating intracellular bacteriophage at any stage during the latent period by arresting phage growth and inducing premature lysis of the infected cells. This is brought about by placing the infected bacteria into the growth medium supplemented with 0.01 M cyanide and with a high titer T6 lysate. It was found in some of the later experiments that the T6 lysate is essential only during the first half of the latent period. Cyanide alone will induce lysis during the latter part of the latent period. Using this method on T4-infected bacteria it is found that during the first half of the latent period no phage particles, not even those originally infecting the bacteria, are recovered. This result is in agreement with the gradually emerging concept that a profound alteration of the infecting phage particle takes place before reproduction ensues. During the second half of the latent period mature phage is found to accumulate within the bacteria at a rate which is parallel to the approximately linear increase of intracellular DNA in this system. However, the phage production lags several minutes behind DNA production. When 5-methyltryptophan replaced cyanide as the metabolic inhibitor, similar results were obtained. The curves were, however, displaced several minutes to the left on the time axis. The results are compared with Latarjet's (16) data on x-radiation of infected bacteria and with Foster's data (18) concerning the effect of proflavine on infected bacteria. Essential agreement with both is apparent.     

Nucleic acid economy in bacteria infected with bacteriophage T2. I. Purine and pyrimidine composition

1. The phosphorus content per infective particle of isolated bacteriophage T2 has been redetermined. It does not exceed 1.8 to 2.2 x 10^–11 µg. The equivalent amount of DNA has been defined in terms of several analytical methods and taken as a unit of measurement of intrabacterial DNA. 2. The DNA of E. coli contains guanine, adenine, cytosine, and thymine in approximately equal amounts, but no hydroxymethylcytosine. One bacterial cell contains 40 to 150 units of DNA, depending on the conditions of growth and the method of measurement. 3. The DNA of phage T2 (one unit per particle by definition) contains guanine, 5-hydroxymethylcytosine, and relatively large amounts of adenine and thymine, but no cytosine. 4. Infected bacteria contain DNA of a composition that varies systematically during the course of viral growth. At all times it resembles a mixture of bacterial and viral DNA. 5. The characteristic bacterial DNA is decomposed after infection, measuring about one-third its initial amount at 20 minutes. The characteristic viral DNA increases in amount, after a short delay, reaching a level of 100 to 400 units per bacterium 30 minutes after infection. At 10 minutes after infection, the two kinds of DNA are approximately equal in amount. 6. The characteristic viral DNA present in infected cells exists in two forms, one consisting of infective particles and one not. The portion not contained in infective particles builds up to 40 to 80 units per cell during the first 10 minutes after infection and afterwards remains roughly constant in amount. Infective particles begin to appear at 10 minutes and account for all or most of the increase thereafter.