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.     

The effect of ultraviolet light on the production of bacterial virus protein

The amount of phage-specific protein in T2-infected bacteria growing in a medium containing radiosulfur, S³⁵, has been studied by measuring the radioactivity in specific antiphage serum precipitates of lysates. In the course of normal infection, non-infective phage antigen has been found to make its first intracellular appearance shortly before the end of the eclipse period, in agreement with the findings of Maaløe and Symonds with phage T4. No such phage antigen is produced either in bacteria infected with UV-inactivated T2 or in T2-infected bacteria whose survival as an infective center has been destroyed by UV irradiation during the early stages of the eclipse period. If the infected bacteria are UV-irradiated only at later stages of the eclipse period however, then phage antigenic protein continues to be synthesized in those infected cells in which DNA synthesis and, a fortiori, production of infective progeny have been almost completely suppressed. It is concluded from these results that once the mechanism for formation of phage-specific protein has been established within the infected cell under the influence of the parental DNA, synthesis of phage-specific protein can continue independently of the synthesis of phage DNA. The possibility that the phage DNA controls the specificity of the phage protein indirectly through substances other than DNA is discussed.     

Evidence that ultraviolet light-induced DNA replication death of recA bacteria is prevented by protein synthesis in repair-proficient bacteria

The ultraviolet light (UV) survival curve of Escherichia coli WP10 recA trp is almost biphasic, with a greatly reduced shoulder but demonstrating a transition to a decreased slope with increasing fluences, indicating the presence in the culture of a low frequency of resistant cells. Treatment of the culture with chloramphenicol before UV exposure brought almost all of the cells to a high degree of UV resistance, by bringing them to the end of their DNA replication cycle. The survival curves of the repair-proficient E. coli WP2 trp showed a similar pattern with chloramphenicol treatment or tryptophan starvation before UV exposure, but only if protein synthesis were blocked by chloramphenicol for 60 min after UV exposure. The results suggest that when recA/lexA-regulon induction is prevented, either by the recA mutation or by inhibition of protein synthesis after UV exposure, death occurs unless the cells are in the resistant state characteristic of bacteria at the end of their DNA replication cycle. With repair-proficient bacteria treated before UV exposure with chloramphenicol, when protein synthesis is not blocked after UV exposure, a marked expansion of the shoulder occurs because of the function of another resistance-conferring mechanism. This mechanism also depends on the recA+ gene since expansion of the shoulder does not occur in recA bacteria when protein synthesis is inhibited before UV exposure.     

Filamentous Bacterial Viruses VII. Inhibition of fd Deoxyribonucleic Acid Synthesis After a Temperature Jump into Protein-Synthesis Inhibitors

Synthesis of fd deoxyribonucleic acid (DNA) was stopped by transferring infected bacteria from 32 C into chloramphenicol or serine hydroxamate at 42 C, but not by addition of these antibiotics at 32 C, and not by a temperature change in the absence of antibiotics. The inhibition of fd DNA synthesis by serine hydroxamate at 42 C was reversed by excess serine. The ability to synthesize fd DNA at 42 C in chloramphenicol was rescued by delaying the addition of chloramphenicol for a few minutes after the transfer from 32 to 42 C. The colony-forming ability of abortively infected bacteria was also rescued from “killing” by delaying the addition of chloramphenicol after a transfer from 32 to 42 C.     

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

Synthesis of Bacteriophage and Host DNA in Toluene-Treated Cells Prepared from T4-Infected Escherichia coli: Role of Bacteriophage Gene D2a

We investigated the synthesis of DNA in toluene-treated cells prepared from Escherichia coli infected with bacteriophage T4. If the phage carry certain rII deletion mutations, those which extend into the nearby D2a region, the following results are obtained: (i) phage DNA synthesis occurs unless the phage carries certain DNA-negative mutations; and (ii) host DNA synthesis occurs even though the phage infection has already resulted in the cessation of host DNA synthesis in vivo. The latter result indicates that the phage-induced cessation of host DNA synthesis is not due to an irreversible inactivation of an essential component of the replication apparatus. If the phage are D2a(+), host DNA synthesis in toluene-treated infected cells is markedly reduced; phage DNA synthesis is probably also reduced somewhat. These D2a effects, considered along with our earlier work, suggest that a D2a-controlled nuclease, specific for cytosine-containing DNA, is active in toluene-treated cells.     

Nucleic acid economy in bacteria infected with bacteriophage T2

1. During the first 10 minutes of viral growth following infection of E. coli by phage T2 in broth, a pool of DNA is built up that contains phosphorus later to be incorporated into phage. This pool receives phosphorus from, but does not contain, the bacterial DNA. 2. After 10 minutes, DNA synthesis and phage maturation keep pace in such a way that the amount of precursor DNA increases moderately for a time and then remains constant. 3. The pool so described is defined in terms of the kinetics of transport of phosphorus from its origins in the culture medium, the bacterial DNA, and the DNA of the parental phage, to the viral progeny. The most interesting parameter of this system is the size of the precursor pool, which measures 10^–9 to 2 x 10^–9 µg. DNA-P (50 to 100 phage particle equivalents) per bacterium. 4. Neither the precursor nor the intracellular phage population exchanges phosphorus with the phosphate in the medium. More interestingly, the phosphorus in mature phage does not exchange with phosphorus in the precursor, showing that maturation is an irreversible process. 5. Maturation is also a remarkably efficient process. About 90 per cent of labeled phosphorus introduced early into the precursor pool is later incorporated into phage. 6. Viral DNA is synthesized at the rate of about 1.5 x 10^–10 µg. DNA-P (7 or 8 phage particles) per bacterium per minute. This is somewhat faster than bacterial DNA is formed, but considerably slower than RNA is formed, in uninfected bacteria. 7. The transport of phosphorus from medium to viral precursor DNA takes an average of 8 or 9 minutes, and from precursor to phage an additional 7 or 8 minutes. 8. Metabolically active RNA has been detected in infected bacteria.     

Control of Bacteriophage-induced Enzyme Synthesis in Cells Infected with a Temperature-sensitive Mutant

The timing of "early" and "late" protein synthesis in Escherichia coli infected with T-even bacteriophage was studied with a temperature-sensitive phage mutant, T4 tsL13. This strain was completely unable to direct the synthesis of phage deoxyribonucleic acid (DNA) at 44 C because it makes a deoxycytidylate hydroxymethylase which cannot act at that temperature. However, the mutant did multiply normally at 30 C. No detectable formation of the late protein, lysozyme, occurred at 44 C, in agreement with the idea, proposed by several workers, that DNA replication is necessary for activation of late genetic functions. However, the formation of an early enzyme, thymidylate synthetase, was shut off at about 10 min, as in normal infection. This implied that separate mechanisms were responsible for cessation of early functions and activation of late ones. That the infected cell at 44 C retained the capacity for synthesis of early enzymes was shown by the fact that DNA synthesis occurred after a culture was transferred from 44 to 30 C as late as 30 min after infection. This synthesis was inhibited by chloramphenicol, indicating that de novo synthesis of an early enzyme can take place at a late period in development. It is suggested that cells infected under normal conditions maintained an appreciable rate of early enzyme synthesis throughout the course of infection.     

Regulation of coliphage f1 single-stranded DNA synthesis by a DNA-binding protein

Control of single-strand DNA synthesis in coliphage f1 was studied with the use of mutants which are temperature sensitive in gene 2, a gene essential for phage DNA replication. Cells were infected at a restrictive temperature with such a mutant, and the DNA synthesized after a shift to permissive temperature was examined. When cells were held at 42 °C for ten or more minutes after infection, only single-stranded DNA was synthesized immediately after the shift to permissive temperature. This indicated that the accumulation of a pool of double-stranded, replicative form DNA molecules is not an absolute requirement for the synthesis of single-stranded DNA, although replicative form DNA accumulation precedes single-strand synthesis in cells infected with wild-type phage. Cells infected at restrictive temperature with the mutant phage do not replicate the infecting DNA, but do accumulate a substantial amount of gene 5 protein, a DNA-binding protein essential for single-strand synthesis. It is proposed that this accumulated gene 5 protein, by binding to the limited number of replicating DNA molecules formed following the shift to the permissive temperature, acts to prevent the synthesis of double-stranded replicative form DNA, thus causing the predominant appearance of single strands. This explanation implies an intermediate common to both single and double-stranded DNA synthesis. The kinetics of gene 5 protein synthesis indicates that it is the ratio of the gene 5 protein to replicating DNA molecules which determines whether an intermediate will synthesize double or single-stranded DNA.     

Reactivation and mutagenesis of UV irradiated lambda phage in bacteria treated with platinum (II) compounds

Treatment of wild type Escherichia coli with cis -Pt(NH3)2Cl2 increased the survival and frequency of clear plaques formation of lambda phage damaged by UV radiation. The reactivation process was present in an uvrA mutant and abolished in a lexA host. Trans-Pt(NH3)2Cl2 and [Pt(dien) Cl]Cl (dien = 2HN-CH2-CH2NH-CH2-CH2-NH2) which, inhibited DNA synthesis less than the cis isomer or not at all, respectively, induced only a slight increase in survival of UV irradiated phage while mutagenesis was not affected. A relation exists between the reactivation of UV damaged phage in bacteria treated with these three compounds and their recently reported abilities to inhibit DNA synthesis and induce recA protein.     

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.     

Inhibitory Effect of Bacteriophage P22 Infection on Host Cell Deoxyribonuclease Activity

Infection of Salmonella typhimurium with phage P22 causes a decrease in the activity of host deoxyribonuclease which degrades single-stranded deoxyribonucleic acid (DNA). This decrease is reversed when the infecting phage is P22c(+); it is not reversed if the infecting phage kills the cell. The decrease does not occur in infections with P22ts25.1 (which only adsorbs and injects DNA) or in infections of a lysogen by a nonvirulent phage. It does occur, however, after infections with other phages which are blocked in phage DNA synthesis. Inhibiting protein synthesis with chloramphenicol does not in itself cause the decrease in uninfected cells, but it does prevent infected cells from showing this effect.     

Excision of Thymine Dimers In Vitro by Extracts of Bacteriophage-Infected Escherichia coli

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from UV-irradiated DNA previously nicked with T4 UV endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 UV-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. A similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7.     

Use of Miracil D to Suppress Bacterial Ribonucleic Acid and Protein Synthesis During Bacteriophage MS2 Infection

Under certain culture conditions, Miracil (35 mug/ml) halts the growth of uninfected Escherichia coli. Cellular ribonucleic acid (RNA) synthesis is almost completely suppressed, whereas deoxyribonucleic acid and protein synthesis are inhibited to a lesser extent. When the drug is added to host bacteria prior to infection with bacteriophage MS2, the phage adsorb to the cells, but penetration of the viral RNA is inhibited. Penetration may be achieved without further viral development by infection in the presence of chloramphenicol. If the bacteria are infected with MS2 in the presence of chloramphenicol, subsequently washed to remove the chloramphenicol, and then treated with Miracil at any time between 0 and 20 min postinfection, a second viral function is inhibited and the yield of progeny phage is reduced. Addition of the drug after 20 min postinfection does not inhibit the infection process. When Miracil is present from early times in infection, only a limited synthesis of both double- and single-stranded virus-specific RNA is observed. The viral RNA species thus produced do not appear to differ from those made in the absence of the drug. A comparison of the activities of the viral RNA synthetase produced during the course of infection in the presence and in the absence of Miracil suggests that a possible cause of the inhibition is the synthesis of an unstable enzyme in the presence of the drug.     

Sulfur-dependent inhibition of protein and RNA synthesis by iron-grown Thiobacillus ferrooxidans

The addition of sulfur to iron-grown Thiobacillus ferrooxidans resulted in a rapid inhibition in the rates of protein synthesis and RNA synthesis. The inhibition of both functions was measured within 15 to 30 min and was maximal between 70 and 90% compared to the iron-grown controls. DNA synthesis, carbon dioxide fixation, and short-term ferrous oxidation rates of the bacteria growing on ferrous ions were not effected by sulfur addition, indicating that the sulfur addition was not perturbing general cellular energy metabolism. The inhibition caused by sulfur mimicked the effect of the RNA synthesis inhibitor, rifampicin, which inhibited both RNA and protein synthesis, but did not correspond with the translational inhibitor, chloramphenicol, which inhibited only protein synthesis in the first hour. Since chloramphenicol pretreatment did not block the sulfur effect, the inhibition of RNA synthesis following sulfur addition was not mediated through protein synthesis.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid I. Protein Synthesis-Dependent Inhibition

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.