- Volume 21, Issue 3, 1959

SUMMARY: In vesicular stomatitis virus inocula containing the transmissible interfering component (T) an exponential relation, at low doses, between inoculum concentration and virus yield suggested that adsorption of one T particle was enough to exclude infective virus. This relation was not maintained at high doses of inoculum, possibly because maximal interference required several hours between adsorption of T and virus particles. An assay method for T, based on the dose which gave 37% of the yield from T-free inocula, showed that the T content of inocula was usually related to passage history and yield of virus on undiluted passage. Treatment with immune serum did not appear to neutralize T, which sedimented in the centrifuge more slowly than did virus. T was much less rapidly inactivated at 56° and by u.v. irradiation than was the infectivity. Despite resemblances between the agent T, incomplete influenza virus and interferon, direct evidence is lacking that T is an incomplete form of the vesicular stomatitis virus or an interferon-like substance.

SUMMARY: The nuclei and small-particle (mitochondria + microsomes) fractions, separated from 510in vitro cultured chick embryo cells during one-step growth of vesicular stomatitis virus, were fractionated into RNA nucleotides by NaCl extraction, hydrolysis and paper electrophoresis. This allowed determination of molar and specific activity ratios of the RNA nucleotides during complementary 32P ‘gain’ and ‘loss’ experiments. Early exponential virus release coincided with a significant increase in uridine content of nuclear and small-particle RNA over uninfected values, and a decrease in specific activity of small-particle RNA uridylic acid in 32P gain experiments. Other fractions showed no change. This suggested a relatively large synthesis of an RNA different from cellular RNA and containing a high proportion of uridine at a time when virus nucleic acid synthesis was probably at its peak. This synthesis appeared to be balanced by a simultaneous breakdown of preexisting RNA.

SUMMARY: A study of the nutritional requirements of Acanthamoeba sp. Neff. has led to the development of a chemically defined medium which contains 18 amino acids, vitamin B12 and thiamin, acetate, citric acid, salts and trace metals. The need for vitamin B12 and thiamin only is an indication that Acanthamoeba may be closely related to the phytoflagellates. Growth of the amoebae in the defined medium is similar to the growth in peptone, but slower. Glucose in high concentration replaced acetate in the denned medium.

SUMMARY: During a stationary phase induced and maintained by the exhaustion of histidine, the total number of histidineless Escherichia coli (h−) remains constant as does the cytological appearance of the cells. If glucose is available to the starved bacteria they die at a rate of c. 10−2 per hr., while mutations to a histidine-independent (h +) condition occur at a rate of c. 10−9 per bacterium per hr. Bacteria adapted to use lactose behave essentially the same way when it, instead of glucose, is available during starvation; but if the starved cells are not fully adapted, death does not occur or is very slow (c. 10−3 per hr.) and the rate of mutation is c. 10−10. When no carbon source is available to the starved cells, mutations cannot be detected.

The following predictions served as tests of the hypothesis of cell-turnover, wherein some bacteria lyse only to be replaced at the same rate by the growth of others— the mutations are presumed to have occurred during this cryptic growth:

1. Mixtures of h− lac+ and h− lac− bacteria in lactose medium where the h− lac+ has a selective advantage should show population shifts if cryptic growth were occurring.
2. During the hypothesized lysis and growth the enzyme β-galactosidase should, at predictable rates, be lost to the medium from adapted cultures in the absence of lactose and developed in unadapted cultures in its presence.
3. Penicillin should kill those cells that grow to replace others, causing an accelerated death and preventing the mutations from taking place.
4. The lysis and death might be microscopically observable on an agar surface.
   
   The hypothesis of turnover did not withstand any of these tests. It was concluded, therefore, that the bacteria under investigation were not dividing and that mutations —genotypic changes (Ryan, 1955 a)—were taking place among them.

Reasons are given to suppose that the mutations result from errors in the replication of genetic material which is in the process of turnover within the non-dividing cells.

SUMMARY: A simple medium is described which allows adequate growth of Vibrio cholerae with the production of only trace amounts of neuraminidase. When added to this medium to give a final concentration of 600 μ m, sialyl lactose or sialyl-N- acetylgalactosamine, two oligosaccharides which contain ketosidically bound N-acetylneuraminic acid and which are substrates of neuraminidase, caused at least a 100-fold increase in the amount of neuraminidase produced during growth of the organisms. N-Acetylneuraminic acid was also found to stimulate neuraminidase production. Galactose and N-acetylgalactosamine had no effect. N-Acetylneura- minic acid was metabolized by V. cholerae as indicated by the disappearance from such enriched medium of substances giving the direct Ehrlich reaction. Evidence is presented which suggests that neuraminidase is an extracellular enzyme.

SUMMARY: Addition of N-acetylmannosamine to a basal culture medium inoculated with Vibrio cholerae strain 4Z stimulated the production of neuraminidase. N-Acetylglueosamine and N-acetylgalactosamine under similar circumstances were inactive. N-Acetylmannosamine, added to medium inoculated with V. cholerae, persisted for at least 12 hr; under similar conditions N-acetylglucosamine was rapidly metabolized. Both D-mannosamine HCl and D-glucosamine HCl resisted enzymic attack by 561V. cholerae. The former compound severely inhibited growth of the organisms; the latter had no such effect. These findings are discussed, particularly in their relationship to the discovery that N-acetylneuraminic acid, which is a condensation product between N-acetylmannosamine and pyruvate, also induces neuraminidase.

SUMMARY: Tobacco rattle virus was isolated from tobacco sap by differential centrifugation; the yield was about 50 mg. virus/1. sap. Purified preparations were highly infective, precipitated optimally at pH 4·0–4·5, and contained one electrophoretic component; chemical analysis and the ultraviolet absorption spectrum suggest they contain about 5 % nucleic acid and 95 % protein. Most rod-shaped particles in purified preparations were 73–77 mμ or 179–192 mμ long: all were 25 mμ wide. These two components were separated by rate zonal centrifugation in sucrose density-gradient solutions: both have the same density in solution as tobacco mosaic virus, and they have sedimentation constants of about 198s and 295s, respectively. No differences were found between long and short particles in serological behaviour, electrophoretic mobility or ultraviolet absorption spectrum; apparently they differ little in gross chemical composition. Only the long particles are infective. Particles slightly shorter than 179 mμ seem non-infective; some of these may be aggregates of two short ones. Short particles rarely aggregate end-to-end at pH 6·8–7·0 but aggregation increases as the pH value decreases.

SUMMARY: The structure of tobacco rattle virus was studied by examining with the electron microscope: (1) shadow-cast mounts of particles partly degraded with alkali, sodium dodecyl sulphate or phenol; (2) unshadowed mounts on thin carbon film of particles treated with solutions of lanthanum nitrate, uranyl acetate, osmium tetroxide, phosphomolybdic acid or phosphotungstic acid.

The particles were tubular, with a central hole of approximately 4 mμ diameter and an outside diameter which varied from 17 to 25 mμ according to treatment. Next to the central hole, which could be filled with lanthanum nitrate or with uranyl acetate, was a region 1–1·5 mμ thick, which stained heavily with osmium tetroxide, phosphomolybdic and phosphotungstic acids. The rest of the particle stained lightly with uranyl acetate, phosphomolybdic and phosphotungstic acids, and showed transverse bands 2·5 mμ, apart. It is suggested that these bands may represent a helical structure similar to that of tobacco mosaic virus, which tobacco rattle virus resembles in many respects.

Some properties of tobacco rattle virus have already been reported (Harrison & Nixon, 1959) and we now describe the structure of the particles as revealed by electron microscopy.

Summary: Preparations made by treating tobacco rattle virus with phenol were about 5% as infective as the initial virus suspensions when assayed on French bean, but less on tobacco. Virus nucleic acid seems to be a major constituent of such preparations. By constrast with whole virus, nucleic acid preparations lost infectivity when incubated for 20 min. with 0·02 mg. ribonuclease/l. or when stored for a day at 20°. Nucleic acid preparations contain threads about 1 mμ in diameter but very few or no virus rods. The infectivity of nucleic acid preparations was little affected by highspeed centrifugation for periods in which more than 95 % of whole virus was sedimented. Ultraviolet-irradiated nucleic acid preparations were photoreactivable although irradiated preparations of whole virus were not. Nucleic acid preparations were only infective when made from purified virus suspensions or from frozen and clarified saps when these contained infective rod-shaped particles 179–192 mμ long. Phenol treatment of purified non-infective particles 73–77 mμ long yielded noninfective preparations.

Summary: A method for the 40-fold purification of glutamic dehydrogenase from Neurospora crassa is described. Strains which are homocaryotic for the alleles am 1, am 2 or am3 produce no detectable glutamic dehydrogenase, but heterocaryons of composition am 1 + am 2 or am 1 + am 3 do possess the enzyme activity. Extracts of am 1 + am 2 mycelia have 10%, or rather less, of typical wild-type activity when assayed at about 20°. Enzyme preparations from am 1 + am 2 are distinguished from wild-type preparations in: (a) their capacity for thermal activation as the temperature is raised between 20° and 35°; (b) their low stability at 60°. Extracts of am 1 + am 3 mycelia show 20–25% of typical wild-type activity. Enzyme preparations from am 1 + am 3 are distinguished from wild-type in their lower affinity for glutamate, and they tend also to be more thermolabile than wild-type enzyme, though less so than am 1 + am 2 preparations. Experiments on mixed enzymes showed no evidence for any effect of either kind of heterocaryon preparation on the properties of wild-type enzyme, or vice versa. It thus seems likely that the effects observed are due to differences in the enzyme molecules themselves. The significance of these observations for theories on the mechanism of inter-allele complementation is discussed.

Summary: Upon the basis of cell-wall composition the oral bacteria Leptotrichia dentium and L. buccalis appear to form a natural generic group related to Nocardia and possibly Lactobacillus. An aerobic oral actinomycete of the type commonly called Nocardia has been shown to resemble closely the anaerobic pathogenic Actinomyces which parasitizes man. The cell-wall composition of Streptomyces, Nocardia, Lactobacillus and Fusobacterium strains is also reported.

Summary: A search has been made for phages active on Clostridium, perfringens, types A, B, C, D, E, and F. Twelve of 49 type A strains, 10 of 31 type B strains, 10 of 26 type C strains and none of either 38 type D, 5 type E or 3 type F strains were lysogenic. The ‘temperate’ phages obtained from these lysogenic cultures, together with similar phages obtained from crude material, such as sewage, were only active on strains of the same type as the lysogenic culture from which they were isolated; some of them were very host-specific. It was possible to induce partial lysis in lysogenic strains by ultraviolet radiation, nitrogen mustard and mercaptoacetic acid.

Other phages isolated from crude material resembled the ‘virulent’ phages of aerobic bacteria. These phages lysed strains belonging to types A, B, C, D, and F.

Despite a long search for phages that would lyse them, a high proportion of the strains of Clostridium perfringens examined remained insusceptible to all the phages isolated. A close relationship between phage susceptibility and colonial morphology was noted. Smooth and rough strains were usually phage susceptible; mucoid strains were usually phage-resistant.

Summary: When young growing filaments or microcolonies of Bacillus anthrads were treated with specific bacteriophage, fragmentation of the filaments followed by complete disintegration of the microcolony were visible microscopically within 2–3 hr. Other Bacillus species were unaffected by the phage. This technique is a simple one for accelerating the identification of Bacillus anthrads.

Summary: Members of the Q1 (A) group of lysogenic strains of Salmonella typhimurium have a common bacterial component and differ only in their prophage content. All are inducible by ultraviolet (u.v.) radiation but, in the cultural conditions which were standard in the experiments here described, never more than. 40% of the bacteria were induced, and this only with exposures which killed approximately 75% of the host strain, Q1. The latent period between irradiation and phage liberation was approximately 45 min. The number of particles per burst varied widely. The most striking difference in the reaction to irradiation of cultures of the different strains was that, without significant variation in the amount of free phage produced, some showed clearing while others did not. Clearing was not directly related to the percentage of bacteria induced, nor was it attributable to the production of a lysin by some strains and not by others. Clearing occurred in strains in which induction took place in bacteria which would otherwise have survived irradiation, and would have enlarged into filamentous forms. In strains in which no clearing occurred, induction took place in bacteria which had received a lethal dose of u.v. radiation, and not in the more lightly irradiated bacteria which were therefore left free to enlarge and so mask the lysis of the induced bacteria. Thus there is evidence that the induction of these closely related prophages is ‘triggered off’ by different stimuli directly related to the degree of damage to the host bacterium caused by irradiation.

Summary: Disintegration of yeast and Clostridium welchii gave cell envelopes which stained Gram-negative by a modified Jensen’s Gram-staining procedure (procedure 1), but stained in part Gram-positive by a modification of the Gram stain in which the films were dried after application of the iodine and before application of the decolorizing agent (90% v/v ethanol; procedure 2). Procedure 2 appeared to give a typical Gram reaction in that a series of organisms gave identical results when stained by the two procedures. Disintegrated Gram-negative organisms gave only Gram-negative material when stained by procedure 2. The Gram-positive material could be removed from the yeast envelopes by extraction at pH 8, but attempts to ‘replate’ the envelopes with the extract were unsuccessful. The results suggest that the Gram-staining reaction of yeast and C. welchii is due in part to a specific staining component and in part to a factor dependent upon the intact nature of the cells. The latter may be associated with the stability or orientation of the staining component or with some membrane effect.

SUMMARY: A mixture of Pseudomonas fluorescens and coliphage T2 was dried at − 20, 0 and + 20° in solutions of each of the 16 combinations of mannitol and sucrose at 0, 0·25, 0·50, and 0·75 m. For P. fluorescens there was no significant average effect of the drying temperature on the numbers which survived drying, but for coliphage T2 average survival increased as the drying temperature was changed from − 20 to 20°. For both organisms, however, there were a number of very highly significant interactions between solutions and drying temperatures. In other words, the effects of the temperature depended on the solutes present and vice versa. There were also some substantial differences between the responses of the two organisms. It is concluded that survival during drying is likely to be determined by factors additional to those studied in these experiments.

SUMMARY: The sporulation of 51 strains of Bacillus megaterium was examined in shaken cultures in potato extract and in Grelet’s defined medium. Some abnormal strains are described in which the spores germinate within the sporangia or very soon after their liberation. The spores of B. megaterium could be completely freed from vegetative cell remnants by treatment with lysozyme followed by thorough washing. Rabbit anti-sera produced with these treated spores did not cross-react with the vegetative organisms. On the basis of their agglutination reactions the spores of 36 strains of B. megaterium were divided into 5 types.

A capsule-like exosporium was detected in wet Indian ink preparations on lysozyme-treated spores of more than half of the 36 strains. The exosporium was rendered visible in the phase-contrast microscope only after the addition of homologous spore antiserum. The exosporium of Bacillus megaterium consists of a capsule-like slimy substance which does not derive from the sporangium but which is probably produced during spore formation. The specific exosporium and spore coat reactions are described.

SUMMARY: Optimal conditions for the adsorption of bacteriophage λ22 by Escherichia coli strain K112 require 0·02 m-MgSO4 and a pH value of the order 6·5. With a multiplicity of input of 20 phage particles per bacterium, 99 % of the bacteria are infected and at least 90 % are lysogenized. Irradiation of the cells with ultraviolet light immediately prior to infection causes them to give the lytic rather than the lysogenic response.