Phage formation in Staphylococcus muscae cultures. IX. Effect of multiple infection on virus synthesis in the absence and presence of specific substrates

1. A strain of S. muscae which requires a substance present in certain acid-hydrolyzed proteins (AHPF) for virus liberation when singly infected in Fildes' synthetic medium no longer needs this substance when multiply infected. 2. In the absence of the AHPF under conditions of multiple infection the amount of phage released is approximately equal to the number of infecting particles between two to ten. Over ten particles per cell has no further effect on the yield of virus. 3. The experimental evidence indicates that it is the phage particle and not some other component in the lysate which can replace the AHPF. 4. The minimum latent period and rise period of cells singly infected in the presence of the AHPF and multiply infected in the absence of the AHPF are the same. 5. The desoxynucleic acid synthesis of cells, infected with a very few virus particles in the presence of excess AHPF and multiply infected with ten particles in the absence of the AHPF, occurs at approximately the same rate, with both infected samples synthesizing about the same amount of desoxynucleic acid and liberating the same yields of virus. 6. A strain of S. muscae which requires aspartic acid for virus synthesis when singly infected does not need this substance when multiply infected, the burst size under the latter conditions depending upon the multiplicity of infection between 3 to 12 particles per cell. 7. The data indicate that the virus released from multiply infected cells in the absence of added AHPF or aspartic acid is newly synthesized virus and not the original infecting particles. 8. The phage particle contains the AHPF and aspartic acid. 9. As a tentative working hypothesis, it is assumed that the AHPF and aspartic acid for phage formation under conditions of multiple infection, in the absence of added AHPF, or of aspartic acid, are contributed by the original infecting particles. 10. Ultraviolet-inactivated phage is adsorbed to the host cell and kills the cell although little virus is released under the experimental conditions. 11. Ultraviolet-inactivated phage particles, if added before the active particle is adsorbed, will greatly inhibit the liberation of new virus particles; but does not do so if added a few minutes after the active particle has been adsorbed. 12. Under the experimental conditions, reactivation of phage when present in multiply infected cells does not occur; and such ultraviolet-inactivated phage cannot serve as a source of the AHPF or aspartic acid, although the AHPF can be liberated from such inactivated particles by acid hydrolysis. 13. The results are discussed in relation to Luria's experiments with ultraviolet-treated phage and to his "gene pool" hypothesis of phage formation.     

Phage formation in Staphylococcus muscae cultures. VIII. Effect of the protein factor and aspartic acid on virus synthesis with various bacterial strains

1. Four strains of Staphylococcus muscae have been isolated which differ in their growth rates and phage syntheses in Fildes' synthetic medium. 2. Two of the strains when singly infected cannot release phage in Fildes' synthetic medium unless a substance present in certain acid-hydrolyzed proteins is added to the medium. One of these strains also requires other substance(s) present in acid-hydrolyzed proteins in order to grow in Fildes' medium. 3. The two strains which do not require the addition of the phage-stimulating factor have been found either to synthesize this substance, or one similar to it. One of these strains will not grow in Fildes' medium unless substance(s) present in acid-hydrolyzed proteins is added to the medium. 4. The purified acid-hydrolyzed protein factor necessary for virus liberation does not affect the multiplication rate of uninfected S. muscae cells in Fildes' synthetic medium. 5. The substance is not needed for the adsorption or the invasion of the host cell by the virus. In the absence of the factor, the virus is adsorbed to the cell and "kills" it. 6. An analysis carried out by means of the one-step growth curve technique has indicated that the substance is not concerned simply with the mechanism of virus release, but is necessary for some initial stage in virus synthesis. 7. With one bacterial strain not requiring the AHPF, aspartic acid had to be present at least during the minimum latent period for the cell to form virus. 8. In the absence of aspartic acid, the virus was adsorbed to the cell and killed it, but no virus was released from singly infected bacteria. 9. If the cells were grown in a medium containing aspartic acid and then resuspended in the medium minus aspartic acid, no virus was released, although such cells contained at least two times the amount of aspartic acid necessary for the burst size in the complete medium. 10. Aspartic acid, a constituent of the virus particle, appears from an analysis of one-step growth curves to take part in the initial phase of phage synthesis. 11. The effect of amino acids on virus formation is discussed in relation to the time sequence of virus protein and desoxyribonucleic acid synthesis.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.     

Growth and phage production of B. megatherium. I. Growth of cells after infection with C phage. II. Rate of growth,phage yield,and RNA content of cells. III. Effect of various substances on growth rate and phage production

I. Lysogenic B. megatherium 899a (de Jong, 1931) produces two types of phage (Gratia, 1936 c) T and C. The T phage forms cloudy plaques and gives rise to fresh lysogenic strains (Gratia, 1936 b) when added to the sensitive strain of megatherium. It may or may not cause lysis, depending on the media (Northrop, 1951). The C phage occurs very rarely) forms clear plaques, does not give rise to lysogenic strains, and causes complete lysis of the sensitive strain under all conditions tested, provided infection occurs. If C phage is added to the sensitive strain, and the mixture allowed to stand, or made into a hanging drop preparation, the infected cells stop growing and lyse completely after 60 to 80 minutes with the liberation of from 50 to 200 phage particles per cell. If, however, C phage is added to a rapidly growing culture of B. megatherium and the suspension shaken at 34°, the cells continue to grow and divide for 50 to 60 minutes, after infection has occurred. They then lyse, with the liberation of from 1000 to 2500 phage particles per cell. II. The following determinations have been made on megatherium sensitive cells growing in 5 per cent peptone at different stages of growth. (1) Growth rate of infected and uninfected cells; (2) RNA, DNA, and protein content; (3) volume of the cell; (4) phage yield per cell by plaque count; (5) phage yield per cell by cell and plaque count; (6) lysis time. The growth rate decreases as the cell concentration increases. The lysis time and the protein N per cell are nearly independent of the growth rate; all the other values increase as the growth rate increases. The ratio See PDF for Equation is nearly constant. RNA and DNA per cell increase less rapidly than the volume, so that NA per unit volume is not constant, but decreases as the size of the cell increases. The phage yield measured under conditions in which the infected cells do not grow (by plaque count) is very nearly proportional to the size of the cell. The phage yield per cell, under conditions in which the infected cells do grow, increases more rapidly than the size of the cells. The phage yield per cell under these conditions may be calculated by the equation See PDF for Equation The determining factor for the variation in phage yield is the growth rate of the cells. This, in turn, is determined by the composition of the medium. III. The growth and phage production of megatherium 899a have been determined in the presence of the following substances: aureomycin, bacitracin, chloromycetin, gramicidin, Merck AB631, Merck AB191, Merck AB624, penicillin, streptomycin, terramycin, tyrothricin, usnic acid, acetone, chloroform, ethyl alcohol, formaldehyde, gentian violet, glycerin, maleic hydrazide, methyl alcohol, phenyl mercuric acetate, sodium fluoride, sulfanilamide, toluene, and urethane. In every case, the lowest concentration of the substance which completely inhibits growth, is also the lowest concentration which completely inhibits phage production. One antibiotic, Merck AB81, causes increased phage production in concentrations which partially inhibit growth, and low phage production in concentrations which completely inhibit growth (as determined by turbidity). Short exposure to ultraviolet light also decreases the growth rate, with increase in phage production. Longer exposure, which completely inhibits growth (as determined by turbidity) results in lysis and phage liberation.     

Ribonucleic Acid Bacteriophage Release: Requirement for Host-Controlled Protein Synthesis

The release of the ribonucleic acid (RNA)-containing phage MS2 from Escherichia coli is accompanied by cellular lysis at 37 C, whereas at 30 C phage are released from intact cells. Chloramphenicol or rifampin prevents the release of progeny phage particles at both temperatures. Neither drug causes an immediate cessation of phage release and after inhibition of protein synthesis by chloramphenicol phage release proceeds for about 17 min at 37 C and about 35 min at 30 C. Rifampin does not inhibit phage release from mutant cells possessing a rifampin-resistant deoxyribonucleic acid-dependent RNA polymerase. The results indicate that a short-lived host-controlled protein(s) is essential for the release of RNA phage particles at both temperatures.     

Phage formation in Staphylococcus muscae cultures. XI. The synthesis of ribonucleic acid,desoxyribonucleic acid,and protein in uninfected bacteria

1. The synthesis of ribonucleic acid, desoxyribomicleic acid, and protein in S. muscae has been studied: (a) during the lag phase, (b) during the early log phase, and (c) while the cells are forming an adaptive enzyme for lactose utilization. 2. During the lag phase there may be a 60 per cent increase in ribonucleic acid and protein and a 50 per cent increase in dry weight without a change in cell count, as determined microscopically, or an increase in turbidity. 3. During this period, the increase in protein closely parallels the increase in ribonucleic acid, in contrast to desoxyribonucleic acid, which begins to be synthesized about 45 minutes after the protein and ribonucleic acid have begun to increase. 4. The RNA N/protein N ratio is proportional to the growth rate of all S. muscae strains studied. 5. While the RNA content per cell during the early log phase depends upon the growth rate, the DNA content per cell is fairly constant irrespective of the growth rate of the cell. 6. Resting cells of S. muscae have approximately the same RNA content per cell irrespective of their prospective growth rate. 7. While the cells are adapting to lactose, during which time there is little or no cellular division, there is never an increase of protein without a simultaneous increase in ribonucleic acid, the RNA N/protein N ratio during these intervals being approximately 0.15. 8. Lactose-adapting cells show a loss of ribonucleic acid. The purines-pyrimidines of the ribonucleic acid can be recovered in the cold 5 per cent trichloroacetic acid fraction, but the ribose component is completely lost from the system. 9. The significance of these results is discussed in relation to the importance of ribonucleic acid for protein synthesis.     

Factors Controlling the Production of Lysogenic Cultures of B. megatherium

(1) The proportion of infected B. megatherium cells which develop lysogenic colonies depends on the number and kind of infecting virus particles and on the culture medium in which the cells are growing. (2) Cells infected with 100 or more T virus particles (from megatherium 899) in yeast extract peptone disintegrate, produce very few virus particles, and less than one lysogenic colony per 10⁷ infected cells. Cells infected with one or a few particles produce 500 to 1000 virus particles each and about 30 lysogenic colonies per 10⁷ infected colonies. (3) T phage obtained from lysogenic magatherium KM cultures produces many more lysogenic cells than does the original megatherium 899 virus. (4) Cells infected with megatherium 899 T virus in peptone medium and then transferred to asparagine medium give rise to 10⁶ lysogenic colonies per 10⁷ infected cells and this transformation will occur even after the infected cells have been in peptone for 60 to 90 minutes and are beginning to produce virus particles. (5) Continued growth of KM strain with either C or T virus from megatherium 899 for several hundred generations in the steady state apparatus results in a lysogenic strain which produces several different types of virus.     

Phage formation in Staphylococcus muscae cultures. VII. Partial purification of the protein factor necessary for virus synthesis

1. A substance is present in autolyzed pepsin solutions which stimulates the release of phage by some strains of S. muscae when added to Fildes' synthetic medium. 2. The substance is assayed by determining the quantity necessary to increase the phage yield to one-half the maximum value, using the one-step growth curve technique. 3. The substance has been concentrated and partially purified (500-fold) by heavy metal precipitation, butyl alcohol extraction, and absorption on norit. 4. No known amino acid or accessory growth substance tested could replace this substance.     

Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).     

The effect of iodoacetate and other inhibitors on phage production and lysis in three phage systems

The minimal bacteriostatic concentration of iodoacetate, azide, or proflavine was added at intervals during the latent periods of virus in three different bacterium-bacteriophage systems (M. aureus, B. mycoides, E. coli). For each interval at which inhibitor was added, the occurrence of lysis and the final yield of phage were determined. In the B. mycoides and E. coli systems, when added during the first part of the latent period, inhibitor prevented lysis and no phage was released. Introduction of inhibitor during the last part of the latent period resulted in normal lysis and in a linear increase in phage that progressively approached the yield obtained in the absence of inhibitor (the later the introduction, the higher the yield). In the M. aureus system, phage production and lysis in the presence of inhibitor followed the same general pattern, except that release of phage and normal lysis occurred in infected cells to which inhibitor had been added quite early in the latent period. Our results, when compared with those of Foster (1948) with proflavine and Bozeman et al. (1954) with chloramphenicol, suggest that (1) the final phage yields represent the amount of mature intracellular virus present at the time of addition of inhibitor and (2) the reactions leading to lysis proceed independently of those leading to the formation of mature virus once phage infection has reached a critical point in time.     

Agrobacterium tumefaciens Conn IV. Bacteriophage PB21 and Its Inhibitory Effect on Tumor Induction

Strain B2 of Agrobacterium tumefaciens Conn produces plaques when seeded against strain B6-806 of the same organism. From such a plaque, a highly virulent bacteriophage was obtained by use of D'Herelle's technique of selecting for virulent phage. On nutrient agar, this phage, PB2₁, produced large clear plaques which did not overgrow. Plaques produced on a glutamate medium and on White's plant tissue culture medium were even larger and in White's medium had a three-dimensional appearance. PB2₁ does not appear to be an oncogenic virus. To the contrary, the addition of phage under circumstances which insure mass lysis completely inhibited tumor initiation. Fewer than 10 phage particles present at the beginning of a 21-hr induction period were able, at times, to inhibit completely tumor induction by highly virulent bacteria (strain B6). The data lend further support to the concept that anything which interferes with the metabolic activity associated with the growth of the bacteria interferes with the tumor-inducing process. Attempts to use the phage to rid crown gall tissue of bacteria were unsuccessful.     

Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

Phage formation in Staphylococcus muscae cultures; the release of the virus from the bacterial cell

1. The release of S. muscae phage in veal infusion medium is correlated with lysis of the host. 2. The release of the bacterial virus in Fildes' synthetic medium occurs in a step-wise manner before observable lysis of the cells occurs. This result has been confirmed by both turbidimetric readings and direct microscopic examination of the infected cells.     

THE GROWTH OF BACTERIOPHAGE AND LYSIS OF THE HOST

1. A new strain of B. coli and of phage active against it is described, and the relation between phage growth and lysis has been studied. It has been found that the phage can lyse these bacteria in two distinct ways, which have been designated lysis from within and lysis from without. 2. Lysis from within is caused by infection of a bacterium by a single phage particle and multiplication of this particle up to a threshold value. The cell contents are then liberated into solution without deformation of the cell wall. 3. Lysis from without is caused by adsorption of phage above a threshold value. The cell contents are liberated by a distension and destruction of the cell wall. The adsorbed phage is not retrieved upon lysis. No new phage is formed. 4. The maximum yield of phage in a lysis from within is equal to the adsorption capacity. 5. Liberation of phage from a culture in which the bacteria have been singly infected proceeds at a constant rate, after the lapse of a minimum latent period, until all the infected bacteria are lysed. 6. If the bacteria are originally not highly in excess, this liberation is soon counterbalanced by multiple adsorption of the liberated phage to bacteria that are already infected. This leads to a reduction of the final yield.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

The B mycoides N host-virus system. II. Interrelation of phage growth,bacterial multiplication,and lysis in infections of the indicator strain of B. mycoides N with phage N in nutrient broth

In nutrient broth at 30 to 32°C., the cycle of virus growth (following adsorption) in lag phase cells of B. mycoides N included a period of intracellular multiplication, ranging from 0.8 to 1.3 hours, succeeded by a sharp rise in the free phage titer and then by a slower rise or a plateau in the extracellular phage content. The yield of virus per infected cell at 30°C., as determined by a modified Burnet dilution technique, was about 76 plaque-forming particles. During the latent period, multiply infected cells showed no change in numbers. Coinciding with phage release, incomplete clearing occurred. The unlysed, remaining cells multiplied and the turbidity rose again. These survivors and their progeny were lysogenic.     

Role of pseudorabies virus glycoprotein gI in virus release from infected cells.

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.     

Bacteriophage T4-Mediated Release of Envelope Components from Escherichia coli

When Escherichia coli B, labeled by prior growth in ¹⁴C-glucose, are infected with T4 phage there is a rapid release of ¹⁴C-nondialyzable material into the medium. About half of this material is derived from the cell envelope as evidenced by its content of phospholipid and lipopolysaccharide and its buoyant density upon isopycnic ultracentrifugation of 1.19 g/cm³. It is similar in its gross chemical and physical properties to envelope material released at a lower rate from growing uninfected cells or from cells whose protein synthesis is inhibited by chloramphenicol (22). The rate of release of this envelope material at a multiplicity of infection (MOI) of 10 is greatest in the first minute after infection, and release is completed by 4 min. The rate of its release, as a function of MOI at 2 min after infection, is greatest at low MOI (e.g., MOI 2 and 4); in addition, the release does not continue above MOI 30. The main conclusion derived from the data is that phage, as part of the process of adsorption and injection of DNA, cause an increased release of envelope substance from the cells. With the assumption that all of the envelope material released is derived from the outer envelope, it is estimated that uninfected cells release 20 to 30% of their outer envelope per hour, whereas infected cells release 30% in 2 min at MOI 30. Further, because release does not continue at high MOI, this phenomenon is not considered to be a direct cause of lysis from without. Data are also presented on the amounts of other non-dialyzable ¹⁴C-components released and on the differences in the kinetics of release from chloramphenicol-treated cells compared to phage-infected cells. To avoid the possibility that the release is due to phage lysozyme which is an adventitious “contaminant” of wild-type phage, a phage mutant (T4BeG59s) devoid of this enzyme was used in these experiments.     

Encephalomyocarditis Virus Ribonucleic Acid Polymerase Associated with 150S Cytoplasmic Particles

Cytoplasmic particles which sedimented at 150S were the smallest structures containing detectable viral ribonucleic acid polymerase in mouse cells infected with encephalomyocarditis virus.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.