E proteins of bacteriophage P22. I. Identification and ejection from wild-type and defective particles.

Of the nine proteins found in the virion of phage P22, four are ejected into the cell after adsorption. The four ejected proteins, termed E proteins, are gp16, gp20, gp26, and gp7. This was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled phage that had been adsorbed to cells and then eluted off the surface with distilled water. Phage particles that lack gp7 (7- particles) or gp20 (20- particles) successfully eject all their E proteins. The 16- particles do not eject gp7. Analysis of phage ghosts showed that they lack gp16, gp20, and gp7, but they have gp26 in close to normal quantities. Our results suggest roles for gp16 and gp26 in DNA and E protein ejection. All four E proteins are possible candidates for roles in helping the phage DNA cross the plasma membrane.     

Non-involvement of lysis during sporulation of Bacillus mycoides in distilled water

Washed vegetative cells of Bacillus mycoides obtained and treated under specified conditions have been found to sporulate when shaken in distilled water under specified conditions. Within limitations of the methods, a heat-resistant cell (spore) is produced for each heat-sensitive vegetative cell present initially. Several different experiments designed to detect massive lysis and cell growth during sporulation in distilled water yielded uniformly negative results. Evidence is furnished for the conclusion that a freshly formed spore (heat-resistant cell) weighs considerably less than its progenitor vegetative cell. The observed results are most satisfactorily explained as a direct conversion of a vegetative cell to a spore.     

The influence of distilled water on the fluidity of protoplasm

Summary It is found that if distilled water is allowed to flow through the leaves of terrestrial plants (Achillea millefolium, Chenopodium album, Rumex acetosella, Ficaria verna), the protoplasm of the cells exhibits increased fluidity. The terrestrial plants thus react in the same way as the earlier investigatedHelodea. The cause is not the absence of nutrient salts or of microelements, since nutrient solutions prepared from distilled water have the same effect. The cause is instead the absence of the fluidity-active substances that have earlier been demonstrated in the soil fluid, and which derive from plants.The effect persists as long as the leaves take up distilled water, but disappears gradually (after 1–3 days) when the uptake has ceased. It is assumed that the return to the normal fluidity value is due to production of fluidity-decreasing substances by the leaf cells. The changes—the increase in fluidity and the subsequent decrease—suggest the existence of a system of equilibrium, which regulates the fluidity value.The expenses of the present investigation were defrayed by a grant from the Science Research Council of Sweden.     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

The effect of sodium chloride on phage formation by staphylococci at elevated temperatures

In experiments with the K strain of Staphylococcus aureus and the K race of bacteriophage suspended in tryptose phosphate broth and maintained at 42 degrees C. it was found that the presence of 1 M NaCl produced certain drastic changes in the relationship between the host cells and the infecting virus: 1. Staphylococci grown at 42 degrees C. in plain broth or in NaCl-broth are "activated," i.e. when growth is stopped by lowering the temperature to 5 degrees C. and phage is added, the activity titre immediately displays a rise of 15- to 16-fold. 2. 1 M NaCl tends to prevent the sorption of phage by cocci and this effect is more pronounced at 42 degrees C. than at 5 degrees C. When the activation test is conducted at 5 degrees C. (the usual temperature) most of the phage is picked up by the cells and the described increase in activity titre follows. If the test takes place at 42 degrees C. there is little sorption and correspondingly little rise in phage titre. 3. Mixtures of staphylococci and phage incubated at 42 degrees C. in NaCl-broth fail to produce phage; the final plaque and activity titres are identical with the initial titres. Here, also, the influence of 1 M NaCl in preventing contact of phage with cocci appears to account for the results. 4. Similar mixtures held at 42 degrees C. in plain broth exhibit a drop of about 60 per cent in activity and plaque titres. The loss of phage may be due to adsorption on dead cells accumulating in the suspension or to the thermolability of the bacterium-phage complex, or to both.     

The B. mycoides N host-virus system. III. Release of phage from the lysogenic strain

Experiments were performed to determine the mechanism of release of phage from the lysogenic strain of B. mycoides N. The results suggest that qualitatively the same situation obtains as in the phage-carrying cultures of B. megatherium 899 and E. coli Li; i.e., the population consists of two kinds of cells: "lysogènes potentiels" and "producteurs." Quantitatively, however, there are more "producers" in a broth culture of the lysogenic B. mycoides N, at least curing the first 4 to 8 hours after cells have been suspended in fresh medium, suggesting that the interaction between host and parasite is one in which the balance is easily swung in favor of the virus. These conclusions are based upon the following lines of evidence: (1) the slow "growth rate" of the lysogenic culture, (2) the fact that the colony count falls far below the plaque count or the filament count (which correspond) for a well washed suspension, (3) the increase in phage output in a large number of tubes, each containing a small number of lysogenic cells, after a few hours' incubation in nutrient broth at 30°C.     

The Effect of Temperature on the Formation of T1 and T2r Bacteriophage

A very rapid variation of yield of T1 and T2r bacteriophage in E. coli at slightly higher than normal temperatures has been observed. T1 phage will develop at 41.2°C but not at 41.7°C. By infecting cells grown on lactose and, therefore, induced to contain beta galactosidase, a technique which indicates when cells have become leaky was worked out. This method shows that at elevated temperatures the enzymatic attack on the cell wall continues to go at a faster rate, while completion of the phage goes more slowly. Thermal constants are given for the processes. Cells at higher temperature, grown on P³² medium develop incomplete particles capable of combining with phage antibody. This suggests that the process affected by the early leakiness of the cells is the completion of the virus protein coat. Supplementing the medium with casamino acids, phosphate, and ATP causes the “rescue” of phage particles by aiding the formation of the coat. This can be achieved several minutes after the cells have become leaky and may form a useful system for the study of phage development in the presence of analogs.     

Dark recovery of UV-irradiated phage T1 I. A minor recovery effect whose exclusion permits the study of survival kinetics under presumably repairless conditions

The survival of UV-irradiated phage T1 is much lower in excision repair-deficient than in excision repair-proficient E. coli cells, due to lack of “host cell reactivation” (HCR). An additional decrease in phage survival occurs when repair-deficient (HCR⁻) host cells have been exposed to UV doses from 3000–10 000 erg mm⁻² of 254 nm UV-radiation prior to infection. The observed effect is attributed to loss of a minor phage recovery process, which requires neither the bacterial excision repair nor the bacterial REC repair system. This type of recovery is little affected by caffeine or acriflavine at concentrations that preclude HCR completely. Its full inhibition by UV-irradiation of the cells requires an approximately 8 times larger dose than complete inhibition of HCR.

In heavily preirradiated cells, the T1 burst size is extremely small and multiplicity reactivation is considerably less extensive than in unirradiated cells. Presumably the survival of singly infecting T1 in these cells reflects absence of any type of repair. The observed phage sensitivity and shape of the curve are compatible with the expectation for completely repairless conditions. The mechanism underlying the minor recovery is not known; theoretical considerations make a phage REC repair mechanism seem likely.     

The effect of various culture media on infection,growth, lysis,and phage production of B. megatherium

B. megatherium cells were grown in various culture media, centrifuged and washed, and suspended in other culture media containing "C" or "T" phage. The per cent of infection, rate of growth, lysis, and phage production were determined. The behavior of the system depends on the culture medium in which the cells were grown and also on the culture medium in which they were mixed with phage. With the T phage it is possible to set up systems which yield the following results: 1. No infection, normal growth, no phage production. 2. Infection, normal growth, no lysis) phage production. 3. Infection, growth for several hours, lysis, and phage production. 4. Infection, no growth, lysis, and phage production. The C phage system is less affected by changes in the culture medium. The change in the behavior of the cells with T phage probably is not due to selection since it occurs without much growth of the culture, and is readily reversible.     

Association of Microcyst Formation in Spirillum itersonii with the Spontaneous Induction of a Defective Bacteriophage

Mitomycin C and ultraviolet light were found to induce the formation of microcysts in Spirillum itersonii. These forms, as well as spontaneously occurring microcysts in this species, were found to contain phage tail parts, rhapidosomes, and a granular substance not seen in normal cells. It is suggested that microcysts are formed as the result of the induction of a defective phage. The production of phage lysozyme within the cell could lead to the formation of spherical forms as the cells lose their structural mucopeptide layer. Complete virus particles were not seen, nor was any biological activity demonstrated when the induced cultures were tested against two other strains of S. itersonii. The other strains of this bacterium also formed microcysts and phage tail parts when induced with mitomycin. Attempts to isolate an organism lacking the defective phage have been unsuccessful.     

Characteristics of a new vibrio-bacteriophage system

A vibrio-bacteriophage system isolated from San Francisco Bay mud is described. The vibrio lyses readily in distilled water and appears to be a new species. In contact with the phage it becomes lysogenic and forms large clumps. The phage presents no unique features.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Chelating Agent Shock of Bacteriophage T5

When two strains of phage T5 (heat-susceptible form T5st(+) and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed "chelating agent shock" (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st(+) was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.     

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.     

Phosphorus utilization by Asterionella formosa Hass

Observations have been made on the relationship in Lake Windermerebetween the growth of Asterionella formosa and the concentrationof dissolved phosphate. Asterionellaformosa has also been grownin culture and the amount of phosphorus required by this organismhas been determined. These experiments have shown that: (1) Asterionella can take up and store in reserve phosphorusfrom concentrations below those found in phosphorus-poor lakes(i.e. below 1 µg.P/l.). (2) Growth can continue in phosphorus-deficient media by makinguse of such reserves, cell phosphorus being steadily reduced. (3) The limiting requirement per cell of phosphorus is veryminute—about 0—06µg P/10⁶ cells/1, so thatinitial concentrations as low as rog.P/l. can theoreticallyproduce a population of some i6 x io6 cells/l. before limitationby phosphorus deficiency. This has been realized in culture.The behaviour of Asterionella formosa growing in nature hasbeen found to conform with that found in culture. It is concludedfrom such observations that phosphorus deficiency is unlikelyto provide a limit to growth of Asterionella in Windermere,despite the very low initial concentration of dissolved phosphate.Further experiments have shown that Asterionella cells low inphosphorus can rapidly take up added phosphate from lake waterbut not from distilled water. Some factors which affect therate of phosphate uptake of depleted cells are investigated,and attempts have ben made to throw some light on the natureof the apparent difference between lake water on the one handand distilled water and a number of artificial lake waters onthe other. No conclusion is reached on the reason why Asterionellacells behave differently in lake water and in artificial media.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.     

Potassium Deficiency-induced Changes in Stomatal Behavior, Leaf Water Potentials, and Root System Permeability in Beta vulgaris L

Studies of the water relations of potassium deficient sugarbeet plants (Beta vulgaris L.) revealed two factors for stomatal closure. One component of stomatal closure was reversible by floating leaf discs on distilled water to relieve the water deficit in the leaves; the other component was reversible in the light by floating the leaf discs on KCl solution for 1 hour or more. Potassium-activated stomatal opening in the light was observed when the guard cells were surrounded by their normal environment of epidermal and mesophyll cells, just as observed by previous workers for epidermal strips. Leaf water potentials, like stomatal apertures, appear to be strongly related to leaf potassium concentration. Potassium-deficient plants have a greatly decreased root permeability to water, and the implications of this effect on stomatal aperture and leaf water potential are discussed. In contrast, petiole permeability to water is unaffected by potassium treatment.     

Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.     

Growth and phage production of lysogenic B. megatherium

Cell multiplication and phage formation of lysogenic B. megatherium cultures have been determined under various conditions and in various culture media. 1. In general, the more rapid the growth of the culture, the more phage is produced. No conditions or culture media could be found which resulted in phage production without cell growth. 2. Cultures which produce phage grow normally, provided they are shaken. If they are allowed to stand, those which are producing phage undergo lysis. Less phage is produced by these cultures than by the ones which continue to grow. 3. Cells plated from such phage-producing cultures in liquid yeast extract medium grow normally on veal infusion broth agar or tryptose phosphate broth agar, which does not support phage formation, but will not grow on yeast extract agar. 4. Any amino acid except glycine, tyrosine, valine, leucine, and lysine can serve as a nitrogen source. Aspartic acid gives the most rapid cell growth. 5. The ribose nucleic acid content is higher in those cells which produce phage. 6. The organism requires higher concentrations of Mg, Ca, Sr, or Mn to produce phage than for growth. 7. The lysogenic culture can be grown indefinitely in media containing high phosphate concentrations. No phage is produced under these conditions, but the cells produce phage again in a short time after the addition of Mg. The potential ability to produce phage, therefore, is transmitted through cell division. 8. Colonies developed from spores which have been heated to 100°C. for 5 minutes produce phage and hence, infected cells must divide. 9. No phage can be detected after lysis of the cells by lysozyme.