Inhibition of lactic streptococcus bacteriophage by crystal violet and other agents

Ninety-nine selected compounds and eleven antibiotic-producing organisms were tested for antiphage activity and host toxicity. A paper disc-agar diffusion method was used for primary screening and quantitative methods were employed for confirmatory investigation. Most of the agents tested, although previously reported as inhibitory to one or more other virus-host systems, did not selectively prevent multiplication of lactic streptococcus bacteriophage. Several compounds which prevented mass lysis were extremely toxic to host bacteria. Crystal violet suppressed growth of two phage strains at a level (1.0 x 10(-7)M) which permitted normal growth of the host cells. Failure of crystal violet to prevent multiplication of many phage strains suggested possible variations in the multiplication mechanisms of different strains of virus. Virustatic levels of crystal violet did not destroy unadsorbed virus, reduce adsorption, or prevent invasion; increase of virus was reduced in one-step growth experiments; mass lysis was prevented or delayed in long time experiments. Addition and removal of crystal violet at various intervals during the latent period resulted in virus yields directly related to the portion of the latent period during which no dye was present. Duration of the latent period was unaffected. Single burst experiments indicated that the yield of plaque-forming particles per infected bacterium was reduced; the proportion of infected bacteria giving rise to active progeny did not appear to be influenced to a significant degree. Crystal violet apparently interferes with intracellular multiplication of the virus, possibly by combination of the dye with phage DNA or fractions thereof at some critical stage in the incorporation of DNA into the virus particle.     

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.     

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.     

Selection for bacteriophage latent period length by bacterial density: A theoretical examination

In bacteriophage (phage), rapid and efficient intracellular progeny production is of obvious benefit. A short latent period is not. All else being equal, a longer latent period utilizes host cell resources more completely. Using established parameters of phage growth, a simulation of three successive phage lysis cycles is presented. I have found that high, but not low, host cell densities can select for short phage latent periods. This results from phage with short latent periods more rapidly establishing multiple parallel infections at high host cell concentrations, whereas phage with long latent periods are restricted to growth within a single cell over the same period. This implies that phage with short latent periods habitually grow in environments that are rich in host cells.     

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.     

Optimizing bacteriophage plaque fecundity

Bacteriophages (phages), the viruses of bacteria, form visible lesions within bacterial lawns (called plaques), which are employed ubiquitously in phage isolation and characterization. Plaques also can serve as models for phage population growth within environments that display significant spatial structure, e.g. soils, sediments, animal mucosal tissue, etc. Furthermore, phages growing within plaques, in experimental evolution studies, may become adapted to novel conditions, may be selected for faster expansion, or may evolve toward producing more virions per plaque. Here, we examine the evolution of the latter, greater plaque fecundity, considering especially tradeoffs between phage latent period and phage burst size. This evolution is interesting because genetically lengthening latent periods, as seen with phage lysis-timing mutants, should increase phage burst sizes, as more time is available for phage-progeny maturation during infection. Genetically shortening latent periods, however, is a means toward producing larger phage plaques since phage virions then can spend more time diffusing rather than infecting. With these larger plaques more bacteria become phage infected, resulting in more phage bursts. Given this conflict between latent period's impact on per-plaque burst number versus per-infection burst size, and based on analysis of existing models of plaque expansion, we provide two assertions. First, latent periods that optimize plaque fecundity are longer (e.g. at least two-fold longer) than latent periods that optimize plaque size (or that optimize phage population growth within broth). Second, if increases in burst size can contribute to plaque size (i.e. larger plaques with larger bursts), then latent-period optima that maximize plaque fecundity should be longer still. As a part of our analysis, we provide a means for predicting latent-period optima-for maximizing either plaque size or plaque fecundity-which is based on knowledge of only phage eclipse period and the relative contribution of phage burst size versus latent period toward plaque size.     

Effect of cyanophage N-1 development on nitrogen metabolism of cyanobacterium Nostoc muscorum

Abstract The impact of cyanophage N-1 development on nitrogenase, glutamine synthetase (GS) and aminotransferases activities in the diazotrophic cyanobacterium Nostoc muscorum was investigated during its latent period. The nitrogenase activity was inhibited after 2 h of infection, suggesting that phage development does not require the product of nitrogenase activity. GS activity was not inhibited until 4 h of infection; however, a decline in activity was subsequently observed. Glutamate oxaloacetate transaminase was inhibited after 1 h of infection and no activity was detectable during the entire latent period. In contrast, glutamate pyruvate transaminase activity increased 2-fold by 4 h of infection and remained higher than the background level until the end of the latent period. The results suggested that under nitrogen fixing conditions, N-1 multiplication proceeds in the absence of nitrogen fixation and that the metabolism of amino acids is altered in favour of phage multiplication.     

Isolation of Streptomyces rimosus Mutants with Reduced Actinophage Susceptibility

The infection of Streptomyces rimosus by the virulent actinophage RP1 was partially characterized. RP1 infection of the host cells results in a dramatic decrease in viable cell count, followed by reduced antibiotic production. Phage-resistant mutants were isolated after mutagenic treatment and RP1 selective pressure. Characterization of the isolated mutants has revealed that RP1 infection had no influence on their growth and antibiotic production. However, multiplication of the phage particles in the lawns of resistant mutants was detected. Since these strains differ from the wild type in RP1 relative efficiency of plating, plaque morphology, and the time necessary for plaque appearance, they are considered to be semiresistant mutants. The propagation of RP1 on semiresistant strains is characterized by lower adsorption of phage particles and longer latent and rise periods. As a consequence, the multiplication of the phage is slower than that of their host, which consequently reduces the ratio of phage to its host, thus diluting out the phage.     

THE INFLUENCE OF RENNET ON BACTERIOPHAGE MULTIPLICATION IN MILK

SUMMARY: Milk inoculated with 1% starter culture and immediately infected with small numbers of phage particles was afforded protection from phage attack for several hours if rennetted within 30 min of infection. The degree of protection was largely dependent upon the multiplication rate of the phage under test. When rennetting was delayed for 90 min after infection, protection was greatly reduced. The effect of early rennetting was to stimulate cell multiplication and retard the increase of phage. This retardation prolonged the period before equality between cell and phage numbers was reached. Even after equality was reached, acid production continued for some hours in milk that had received early addition of rennet.     

THE INFLUENCE OF ACIDITY ON THE MULTIPLICATION OF LACTIC STREPTOCOCCAL BACTERIOPHAGE IN LIQUID MEDIA

SUMMARY: Mass lysis of lactic streptococci infected with baeteriophage at 30° was prevented at pH 5·10. At lower pH values no multiplication of phage followed infection, and prolonged incubation at 30° resulted in loss of phage particles from unlysed samples. Adsorption of phage particles on host cells was unaffected by acidity, but no phage penetration of host cells took place. Host cell properties were apparently unchanged by adsorption of phage particles in acid whey.     

The isolation and characterization of a temperate phage, Y46/(E2), from Erwinia herbicola Y40.

A temperate phage was induced from exponential phase cells of Erwinia herbicola Y46 by treatment with mitomycin C. The phage was purified by single plaque isolation, and produced in bulk by successive cultivation in young cultures of E. herbicola Y 178. Phages were concentrated from culture filtrates by rate zonal centrifugation and resuspension in 0.02 M Tris buffer, pH 7.2, twice, yielding suspensions of about 5 times 10(11) PFU/ml. Purification was achieved by centrifugation in buffered sucrose solutions. The band at the 30/40% sucrose interface yielded intact particles having regular hexagonal heads and lonb contractile tails, with base plates. Fibers were not seen. The mean dimensions were head, 51 nm; neck length, 11 nm; overall tail length, extended, 98 nm and contracted, 75 nm; diameter of tail sheath, 24 nm. The phage was stable from pH 4.0 to 11.0, but unstable at pH 3.0, the response being independent of the suspending medium used. At pH 3.0, a survival curve having biphasic appearance was observed, which was not due to a mixed population of phages. Stability to heat was good up to 45 degrees C, above which a logarithmic decline with temperature increase occurred. The average inactivation rate constant at 50 degrees C and pH 6.8 was 0.15 min-1. Adsorption to E. herbicola Y 178 cells exhibited first-order kinetics, the adsorption rate constant being 2.5 times 10(-10) ml/min. One-step growth-curve experiments indicated a burst size of 35-40, and a minimum latent period of 80 min. Probit analysis gave a mean latent period of 140 min (SD 25). The phage caused lysis of only E. herbicola strains Y178 and Y186.     

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.     

Isolation and partial characterization of a bacteriophage active on Hyphomicrobium sp. WI-926

Isolation of a Hyphomicrobium phage from raw sewage from Athens, Ohio, was achieved by a combination of differential centrifugation, filtration, enrichment in mixed Hyphomicrobium cultures, and purification on individual host strains by subculturing single plaques in soft agar overlayers. Enrichments with water from Lake Erie and Lake Beechwood (Ohio) were unsuccessful. Out of 21 Hyphomicrobium strains and 22 other Gram-negative and Gram-positive bacteria tested, only Hyphomicrobium WI-926 (isolated from a German forest pond) was susceptible. This phage had an isometric head (diameter between opposite apices, 67 nm) and a short (12 nm), noncontractile tail and belongs thus to the morphogroup C1. It contained double-stranded DNA. The single-step growth curve showed a latent period of 9 h, a rise period of 6 h, and a burst size of 35. The various differentiation stages in the host development exhibited different affinities for phage adsorption and development. While all stages allowed phage adsorption, the daughter cells were most efficient. Phage multiplication was limited to daughter cells, and the development of infected swarmer cells was arrested permanently at this stage.     

Intracellular growth of bacteriophage studied by roentgen irradiation

Growing Escherichia coli infected with bacteriophage T2 was x-rayed during the 21 minute latent period which elapses between infection and lysis of the cells. Survival curves of the infected bacteria were determined almost from minute to minute; they disclosed the following facts which are related to the process of phage growth: During the first 7 minutes, the infective virus particle remains in the cell unique and genetically intact. The host cell synthesizes some ultraviolet-absorbing material probably devoted to building future particles. From the 7th to 9th minute the x-ray resistance of the virus particle increases, probably because of some internal change. Then, multiplication starts and is completed at about the 13th minute, when an average of 130 virulent units is present per cell, displaying an x-ray resistance twice as high as that of the extracellular virus particle. From 13 minutes to the end, the new units progressively recover the x-ray sensitivity of the extracellular virus. Nothing can be said about either the rate of multiplication between 9 and 13 minutes, or the nature of the multiplying units, except that they are more radiation-resistant (probably smaller) than the extracellular virus. The first steps of the growth process are favored by an unknown component of the lysate, different from the active particles. Several particles can grow in the same host cell.     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

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.     

Mutations in coliphage p1 affecting host cell lysis

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.     

Phage Typing Reactions on Brucella Species

The nature of the phage typing reactions on Brucella species was determined by rates of adsorption and infection, one-step growth experiments, and susceptibility to lysis from without. The highest rates of adsorption and infection were obtained on smooth B. abortus cultures, and large clear plaques were produced. One or a few phage particles per B. neotomae cell killed about one-half of the cells, but some went through an infective cycle and released mature phage that resulted in production of small clear plaques. With B. suis, more phage particles per cell were required to kill, replication did not occur, and plaques were not observed. Still greater numbers of phage particles were required to cause some inhibition of growth of B. melitensis lawns. Rough Brucella cultures and species, such as B. ovis and B. canis, were not affected by the highest concentrations of phage. B. abortus cultures of intermediate colonial morphology adsorbed phage, but only a few infected cells (after a delayed latent period) released mature phage. An infected culture or colony appeared normal until spontaneous phage mutants appeared which could penetrate the cell wall more effectively than the parent phage. The mutant phage multiplied more rapidly, and the colony changed to a sticky white form.     

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.