- Volume 38, Issue 1, 1965

The inactivation of foot-and-mouth disease virus and vesicular stomatitis virus by long-lived inactivating agents in X-irradiated buffer solutions was investigated and compared with the findings of Alper (1952, 1955, 1956) relating to the inactivation of bacteriophage by similar agents. A comparison is made between the effects of these inactivating agents and of hydrogen peroxide to which there is a resemblance. Possible reaction kinetics are discussed in an attempt to explain the form of the survival curves resulting from such inactivation reactions. The radiation sensitization of the viruses to these modes of inactivation is also described and discussed.

Chlorella vulgaris grown with nitrate as nitrogen source had high nitrate reductase activity. During nitrogen starvation the rate of nitrate assimilation increased but the specific activity of nitrate reductase in extracts decreased sharply; a similar decrease occurred after transfer to an ammonium medium. Organisms grown with ammonium as nitrogen source did not assimilate nitrate but acquired the ability to do so after nitrogen starvation; nitrate reductase activity in these organisms was initially very low but increased during nitrogen starvation and then decreased. After 18 hr of nitrogen starvation ammonium-grown and nitrate-grown organisms assimilated nitrate rapidly at a rate some ten times greater than the activity of nitrate reductase in cell-free extracts would allow. The specific activity of glutamic dehydrogenase was about 40% higher in extracts from ammonium-grown organisms than in extracts from nitrate-grown organisms and its activity increased by 40-100 % during nitrogen starvation. The activity of malic dehydrogenase was unaffected by 18 hr of nitrogen starvation. The activity of isocitrate lyase decreased by about one-quarter when acetate was present but decreased much more markedly when the organisms were nitrogen-starved in the presence of glucose.

The molar growth yield of Aerobacter aerogenes growing anaerobically with glucose in a mineral medium was almost doubled when NO3 − was added as hydrogen acceptor. About half a mole of NO3 − was reduced to NH4 + per mole of glucose. The amount of ATP produced from glucose fermentation calculated from the molar growth yield and the acetate production was about 3 moles ATP/mole glucose. The total amount of ATP produced, deduced from the molar growth yield, was used to calculate that about 3 moles ATP were produced per mole of NO3 − reduced. Comparable experiments with mannitol as carbon source gave the same results. When nitrite was used as hydrogen acceptor the anaerobic molar growth yield was the same or even smaller than in its absence, although the acetate production increased. The results suggest that during NO2 − reduction no ATP is produced and that the ATP formed during reduction of NO3 − to NH4 + is completely produced during the reduction of NO3 − to NO2 −.

Twelve phages isolated from sewage and active on lactobacilli were examined by electron microscopy with a negative-staining technique. Those phages active on Lactobacillus fermenti (heterofermentative) possess icosahedral heads and sheathed tails which end in base-plates and pins. Those phages active on Lactobacillus casei (homofermentative) differ in that their heads are octahedral or icosahedral and they possess collars. The overall length of all the phages is similar and their base-plates remain attached to the sheaths when these contract. No tail fibres were seen. A temperate Lactobacillus fermenti phage was also examined. It has a small hexagonal head and a long unsheathed tail which ends in a star-shaped structure.

Soluble material extracted from purified vaccinia virus particles produced eight precipitin lines with antiserum by immunodiffusion. The material contained about 20 % of the viral nitrogen and was obtained by extraction with alkaline buffers up to pH 10·5, by prolonged autolysis or, more conveniently, by digestion with trypsin. It appeared to originate mainly from the virus surface but, as it also contained about 5 % of the viral DNA and a small proportion of the virus particles were converted to ghosts, some of the precipitin lines probably represented internal components of the virus. When more material (up to 75% of the viral-N) was extracted no further precipitin lines were detected but some immuno-specificities may have been destroyed. Serial extractions at increasing alkaline pH values separated the antigens from most of the viral DNA and indicated that the ‘nucleoprotein antigen’ of earlier workers was probably an artifact.

Soluble antigens extracted from rabbit skin infected with vaccinia virus produced immunodiffusion patterns containing up to 17 lines. At least 5 of the components which produced lines were labile when heated at 60°. Soluble material obtained from purified vaccinia virus particles produced 8 precipitin lines and an additional virus component was detected with antiserum prepared against inactivated virus particles. Seven of the virus particle precipitin lines were identical with soluble antigen lines, but up to 10 of the soluble antigen lines did not appear to represent components of the virus particle. It is suggested that they represent specific substances (perhaps enzymes) required for virus replication but not incorporated into the virus.

The concentration of phospholipids in pigmented Rhodopseudomonas spheroides was higher than that in organisms lacking the photosynthetic pigments; the amounts of lipid phosphorus/mg. protein were, respectively, 157 and 90 mμmoles. Phosphatidylethanolamine comprised about 40% of the phospholipids, the remainder consisting of phosphatidic acid, phosphatidylglycerol and phosphatidylcholine. The phospholipids were confined to the membrane fraction of the organisms. As organisms adapted from the non-pigmented to the pigmented state the amount of lipid phosphorus/mg. protein increased. Experiments with radioactive phosphate showed phosphatidyl glycerol to incorporate label more rapidly than the other phospholipids. It is suggested that the formation of the photosynthetic pigments may be accompanied by modifications to the membrane structure so that it can accommodate more phospholipids.

The synthesis and breakdown of long chain polyphosphate in Chlorobium thiosulfatophilum was studied. An enzyme is present which reversibly transfers phosphate groups from polyphosphate to adenosine diphosphate to give adenosine triphosphate. A second enzyme, an adenosine triphosphatase probably located in the cytoplasmic membrane, hydrolyses the adenosine triphosphate so formed to adenosine diphosphate and ortho-phosphate.

Hexokinase was determined in extracts of Escherichia coli by measurement of the reduction of nicotinamide-adenine dinucleotide phosphate by glucose-6-phosphate dehydrogenase, and the glucose-6-phosphate formed by the action of hexokinase. This method gave reproducible results. Extracts of E. coli contained low hexokinase activity when the organism had been grown in the absence of glucose and this enzyme activity was increased after adaptation of the organism to growth on glucose. The E. coli needed 2–2·5 divisions to reach the degree of hexokinase activity of glucose-grown bacteria when transferred to salts + glucose from media not containing glucose.

Cocoa yellow mosaic virus from Sierra Leone is readily transmitted by sap to many woody and herbaceous dicotyledonous plants. It is serologically related to wild cucumber mosaic virus and to turnip yellow mosaic virus, and has similar chemical and physical properties. It is stable when frozen or lyophilized, inactivated when kept for 10 min. at 65°, and is precipitated without loss of infectivity in a half-saturated ammonium sulphate solution. The particles appear to be 25 Mμ across when mounted in neutral phosphotungstate, and 29 Mμ when shadowed. All preparations contained infective nucleoprotein particles which sedimented at 108 S, and non-infective protein shells which sedimented at 49 S.

Sixteen serological types of Mycoplasma previously distinguished by complement-fixation tests were compared by means of a gel precipitin (Ouchterlony) technique. Each of the sixteen gave a consistent pattern of three to seven precipitation lines with its homologous antiserum. Cross-reactions occurred between heterologous strains, especially with antisera produced with the aid of an adjuvant. Homologous reactions were complex and sufficiently distinctive for the technique to be used to identify unknown strains. Two of the sixteen types, Mycoplasma mycoides var. mycoides and M. mycoides var. capri, had certain antigenic components in common. The appearance of other cross-reactions suggested that the majority were due to the presence in different species of partly related rather than identical antigens.

The growth of Brucella abortus, B. melitensis and B. suis in laboratory media was inhibited by several analogues of erythritol. The use of erythritol analogues in this manner was suggested by previous work on the localization of brucellas in brucellosis. The growth of B. abortus within bovine phagocytes was inhibited by analogues of erythritol under conditions in which streptomycin was ineffective. Although selected erythritol analogues did not eliminate brucellosis in guinea pigs, they produced a significant diminution in infection and showed no toxicity to the host.

The growth of a virulent strain of Brucella abortus was stimulated by low concentrations of erythritol in a medium containing high concentrations of glucose and a wide range of amino acids. During growth in this medium the organisms used about 1 1/2 times their weight of erythritol as a carbon and energy source. The effect of erythritol was specific, since several C3 to C6 homologues had no growth-stimulating activity. Radiotracer studies snowed that a large proportion of the carbon of the erythritol was excreted as carbon dioxide; that remaining in the organisms was fairly uniformly distributed over all components. Erythritol and 2-deoxy-2-fluoro-DL-erythritol inhibited incorporation of glucose by the organism.

An aerobic actinomycete isolated from a type of periodontal disease in hamsters showed a characteristic filament/diphtheroid dimorphism. Incubation under carbon dioxide favoured development of the diphtheroid form, whereas aerobic incubation favoured the mycelial form. Iron depletion also induced filament formation. Although magnesium concentration directly affected the rate and amount of growth, no effect on morphology was observed. Critical concentrations of streptomycin sulphate and chloramphenicol also induced mycelium formation.

Growth, respiratory activity and the ability to accumulate D-glucosamine were examined in three psychrophilic and three related mesophilic micro-organisms at temperatures between 0° and 20°. Each of the psychrophils (strains of Arthrobacter, Candida and Corynebacterium erythrogenes) grew well at 0° and also respired exogenous glucose and accumulated glucosamine at this temperature. The minimum temperatures for growth (5-10°) of the mesophilic strains of Arthrobacter and Candida utilis were approximately the same as those at which the organisms ceased to respire glucose and accumulate glucosamine. But the mesophil Corynebacterium xerosis respired exogenous glucose and accumulated glucosamine at temperatures as low as 10°, which is well below the minimum temperature for growth of this bacterium in freshly inoculated culture (20°). Cultures of C. xerosis transferred from 30° to 15° in the mid-exponential phase of growth were capable of a limited amount of growth at the lower temperature, corresponding approximately to a doubling in the size of the population; late-exponential phase cultures when transferred to 15° did not have this ability. Although cultures of C. xerosis transferred from 30° to 10° did not grow, the bacteria were able to respire exogenous glucose and accumulate glucosamine even after 48 hr at the lower temperature. The psychrophilic organisms did not differ from their mesophilic counterparts in their sensitivity to 2,4-dinitrophenol, uranyl ions and nystatin, three metabolic inhibitors which affect respiratory activity and sugar uptake in microorganisms.

Rutilantin A has been tested for antiviral activity in vitro. This antibiotic had no demonstrable effect on the growth of poliomyelitis or influenza viruses, but it completely inhibited the multiplication of rabbitpox virus. It was effective against rabbitpox virus when added to tissue-cell cultures as late as 4 hr after infection. The lowest concentration of rutilantin that prevented the formation of infective rabbitpox virus didnot apparently affect the synthesis of viral DNA or soluble antigens. It is concluded that rutilantin inhibits a late stage of virus development. The toxicity of rutilantin for both cultured tissue cells and animals makes it unlikely to be of value in the treatment of virus disease.

Free and esterified cholesterol were determined in the lipid extract of nine parasitic and saprophytic Mycoplasma strains. The saprophytic strains were usally free from esterified cholesterol. Esterified cholesterol was found in the parasitic strains at a ratio of 1:8 to 1:3 to free cholesterol; in the growth medium the ratio was about 4:1 [4-14C]-cholesterol added to the growth medium was incorporated by all Mycoplasma stains without esterification or any other chemical change. [4-14C]-cholesteryl oleate was incorporated into the lipid of the parasitic strains and was not hydrolysed to yield free cholesterol. The small amounts of radioactive oleic acid incorporated by serveral Mycoplasma strains during grwoth was not detected in the cholesteryl ester fraction. Our results show that under the grwoth conditions examined, the Mycoplasma organisms tested were unable to esterify cholesterol or hydrolyse cholesteryl esters.