- Volume 11, Issue 2, 1954

SUMMARY: The investigation of the metabolism of thiocyanate by pure cultures of Thiobacillus thiocyanoxidans was complicated by the fact that thiocyanate serves both as source of energy and as source of carbon and nitrogen. It was, therefore, difficult to separate oxidation from carbon dioxide fixation. The equation which best represented the resultant of these two processes was:

2KCNS + 5H2O + 3O2 = K2SO4 + (NH4)2SO4 + CO2 + (CH2O).

Thiocyanate was first hydrolysed to cyanate and sulphide. Cyanate was further hydrolysed to carbon dioxide and ammonia and sulphide was oxidized to sulphate. The oxidation of sulphide approximated to the equation:Na2S + 2O2 = Na2SO4. Gas uptakes were lower than would be expected from this equation, although special precautions were taken to prevent loss of sulphide.

Thiosulphate was oxidized to sulphate according to the equation:

Na2S2O3 + H2O + 2O2 = Na2SO4 + H2SO4.

Fixation of carbon dioxide after the oxidation of thiosulphate was shown, but the efficiency of energy utilization appeared lower than during the oxidation of thiocyanate.

Thioacetamide and thioacetic acid were oxidized to sulphate but this was believed to be preceded by a chemical breakdown to sulphide. Sulphite, metabisulphite, dithionite, dithionate, tetrathionate, trithionate, thiourea and cysteine were tested; no evidence was obtained of their utilization.

SUMMARY: A number of cultures of chitin-decomposing aerobic bacteria were isolated by the enrichment technique. Chitin decomposition was followed by measurement of loss of alkali-resistant insoluble substrate. Agitated submerged cultures secured a much more rapid breakdown of chitin than static cultures. The exocellular chitinase produced by a species of Streptomyces in agitated submerged culture was studied. The effects of pH value and substrate concentration on enzyme activity were examined and the results used to establish a chitinase assay in terms of reducing-sugar production. The enzyme was an inducible one (adaptive). The enzyme was concentrated by ultrafiltration and lyophilization. The water-soluble reducing materials produced by the enzyme from chitin were identified as N-acetylglucosamine and its corresponding disaccharide, N.N-diacetylchitobiose.

Hydrogen sulphide production by washed bacterial suspensions in buffered substrate solutions was examined. The suspensions were tested for H2S production from cysteine, cystine, homocystine, methionine, mercaptoacetate, sulphite, sulphate or thiosulphate. Some strains of the Proteus, Klebsiella, Salmonella, Arizona and Bethesda groups produced hydrogen sulphide when 1011 or more washed organisms/ml. were incubated at 37° without an added source of sulphur. Generally tenfold greater quantities of bacteria were required to produce hydrogen sulphide within 24 hr. from homocystine, sulphite or thiosulphite as compared with cystine or cysteine. For the production of hydrogen sulphide from sodium mercaptoacetate, 10- and 100-fold larger quantities of bacteria were required. In the Brucella group all strains behaved alike, except two non-smooth strains which were spontaneously agglutinated in the buffer + substrate solutions and did not produce hydrogen sulphide. Among the Enterobacteriaceae the most active strains belonged to the Proteus group, the most inactive strains to the Large-Sachs group. All strains of the Arizona and Bethesda groups, and some Klebsiella, Salmonella, Serratia and Providence strains multiplied on cysteine and cystine + buffer solutions, utilizing for growth the split products, pyruvic acid and ammonia.

Hydrogen sulphide production was inhibited by penicillin or streptomycin. Non-multiplying micro-organisms exposed to penicillin, streptomycin, aureomycin or chloramphenicol for 24 hr. at 37° remained viable, but lost their ability to produce hydrogen sulphide. This ability was regained on subculture. The hydrogen sulphide production of bacteria inactivated by penicillin was restored when the cells were removed by centrifugation, and residual penicillin destroyed by penicillinase. Addition of bacterial extracts heated at 50° or pyridoxal phosphate, separately or together to inactivated bacteria, reactivated the hydrogen sulphide production of Proteus vulgaris. Bacterial extracts heated at 100° exerted slight reactivating effects.

The fine filaments of Bacillus megaterium are shown to arise from large basal cells: they have paired nuclear bodies within walls differing in composition from those of the normal vegetative cells. No evidence was found for any sexual fusion of these filaments.

A diazotizable amine and hypoxanthine accumulate in cultures of Yeast 47 grown in a defined medium containing added methionine and suboptimal biotin. Amine accumulation is depressed by the presence either of added adenine, or of certain amino acids, of which aspartate, norleucine, norvaline and threonine are the most effective. The amine differs from 4-amino-5-imidazolecarboxamide and the corresponding amidine. Spectrophotometric measurements suggest that the amine lacks the completed 6-membered ring structure of the purine skeleton, but it has biological activity in lieu of adenine for the adenine-requiring Yeast 19, and of guanine for a strain of Lactobacillus brevis.

Hypexanthine substitutes for adenine in the growth of Yeasts 19 and 47, and for adenine + guanine in Lactobacillus brevis, which suggests that hypoxanthine is a bio-precursor of both adenine and guanine. The accumulation of hypoxanthine in cultures of Yeast 47 may thus be the result of a synthetic block, conditioned by biotin deficiency, which prevents the conversion of hypoxanthine to adenine and guanine. The possible relationships between diazotizable amine and hypoxanthine are discussed.

Chamberlain, Cutts & Rainbow (1952) reported the conditions under which Yeast 47 formed pigment and accumulated a diazotizable amine in liquid medium. This paper reports a study of the constituents present in amine-containing concentrates prepared from culture fitrates of Yeast 47.

Paper chromatographic methods were used to determine the amino acids in unhydrolysed corn steep liquor and to study the uptake and synthesis of amino acids by Penicillium chrysogenum. Of the eleven amino acids found in corn steep liquor all were taken up by the organism prior to the main period of penicillin formation. Only traces of ninhydrin-reacting material were present in the later samples of medium.

Glutamine, in addition to the eleven amino acids detected in corn steep liquors, was found in mycelium grown on medium containing corn steep liquor. Mycelium from Penicillium chrysogenum grown on medium containing inorganic nitrogen as sole nitrogen source showed the presence of the same twelve amino acids.

A new species of Cryptococcus has been isolated from six of eight soil samples taken in the province of Otago, New Zealand. It is distinguished from other species of the genus by its ability to utilize glucose, maltose, lactose, galactose and potassium nitrate, but not sucrose.

Various methods of preparing microcultures with liquids (hanging-drop) and solids (collodion, cellophan, agar) were compared with respect to the mode of growth of the test organism on a complex nutrient substrate. Cell division was promoted in general by lower temperatures, by ageing, by lack of humidity in solid cultures, by the action of surface tension operating in the more convex droplets, and by the addition of surface-active agents to liquid media. By varying the carbon sources in chemically defined media it was found that starch, glycogen, and to some extent glucose, favoured filamentation, i.e. retarded or inhibited cell division, in comparison with a considerable variety of other sugars. Of the nitrogenous sources, nitrate resulted in relatively poor growth mainly composed of mycelial aggregates that later exhibited cell division to the extreme limits; ammonium salts yielded more profuse filamentous growth with less subdivision; certain single amino acids induced a more uniform development of short rods: while casein hydrolysate commonly produced a variety of cell elements showing subdivision in all stages. Single cell isolates were used throughout, and the mode of growth was studied by means of phase-contrast and electron microscopy.

A cultural description of the test organism, Nocardia turbata, n.sp. is given at the end of the paper.

SUMMARY: The methods described allowed successful cultivation of 60 % of single cell isolations in the case of an aerobic, transiently chain-forming and polymorphous organism; 40 % in the case of an organism of related morphology and similar ready viability, but presenting technical difficulties because of the lipophilic nature of the surface membranes; and approximately 2 % in the case of a micro-aerophilic or anaerobic organism of restricted viability.

SUMMARY: When Aerobacter aerogenes was harvested from citrate + mineral salt media in which the concentration of carbon source limited the crop, citridesmolase activity of cells was high but was absent when growth ceased with citrate in excess. These differences were abolished by disintegration of the cells. The ability of Escherichia coli to dissimilate citrate was measurable only after growth in a medium containing peptone, citrate and glucose: when the latter was consumed activity fell and was revived by further glucose additions. Differences in activity of such cells were not abolished by their disintegration. Cells lost their activity when incubated for 30 min. with phosphate buffer or with the citrate + mineral salt medium that supports growth of A. aerogenes. E. coli grown at the expense of glucose in a mineral salt medium + citrate, did not utilize citrate for growth during the first day and no citridesmolase activity could be detected, but when subsequently tested in a medium which permitted development of activity, higher activities were reached by cells that had grown in presence of citrate. E. coli grown in peptone + citrate + glucose medium possessed little oxaloacetate decarboxylase; by the action of disintegrated cells, citrate gave rise to equimolar quantities of keto-acid, chiefly oxaloacetic acid.

SUMMARY: Lumpy skin disease virus in allantoic fluid was found to be stable when subjected to wide variation of hydrogen-ion concentration under differing conditions of time and temperature. The virus could be concentrated by adsorption on the precipitate formed when allantoic fluid was dialysed at pH 4·s5; adsorption on calcium phosphate was also demonstrated. Preliminary purification of the virus could be effected by these methods.

SUMMARY: Two strains of Neurospora crassa, which require α-amino nitrogen for growth as a result of a single gene mutation (am), lack glutamic dehydrogenase. Extracts of mutant mycelium contained no detectable enzyme, although as little as 0·2 % of the amount normally present in the wild type should have been detectable in some experiments. Mutant extracts contained no detectable enzyme inhibitor. A balanced heterocaryon containing am nuclei and nuclei carrying an unrelated mutation produced less enzyme than did wild type mycelium, but there was no indication that the am nuclei were suppressing the activity of the non-am nuclei in promoting enzyme production. The production of the enzyme by the wild type was not strikingly dependent on conditions of growth although the addition of glutamate to the medium seemed somewhat to depress the enzyme concentration. This effect was more marked when glutamate was supplied as a sole nitrogen source. The mutation had little or no effect on the concentrations of two other dehydrogenases or of several transaminases, all related to glutamic dehydrogenase in their substrate specificities. The possible significance of these observations is discussed.

SUMMARY: A tissue grinder is described, consisting of a nylon pestle mounted on a stainless steel shaft and revolving within a Pyrex tube; a screw is cut in the pestle to increase its efficiency. The whole device is sealed with a Subaseal bung and may be sterilized by autoclaving. Optimum grinding with minimum heating is produced by rotating the pestle at 500 r.p.m. with a small electric motor.

SUMMARY: Further recombination data have been obtained from matings involving different F+ auxotrophic mutants of Escherichia coli strain K-12 with the same K-12 F− auxotroph. Variations in the distribution of sugar fermentation markers among the progeny have been explained by the influence of the ‘contra-selected’ F+ auxotrophic marker. It is suggested that the data supports the conception of a single linear chromosome in K-12, which fragments randomly during recombination.

SUMMARY: The adaptation of viruses to attack new hosts which originally are resistant greatly widens the sphere of influence of viruses on bacterial variation and evolution. The viruses attacking diphtheria bacilli are highly specific in their host range activity but many of them are readily adapted to lyse strains of other serological types. The range of adaptability of viruses is largely unknown and studies on the specificity and adaptability of bacterial viruses are complicated by the phenomenon of autoadaptability. Although virus-carrying bacterial strains are normally resistant to the viruses with which they are symbiotically infected it is now found that in many cases the viruses can be readily adapted to attack and lyse the parent bacterial cells. This has been found to occur spontaneously in cultures, and accounts for many cases of bacterial variation and dissociation. The existence of closed evolutionary chains in Iysogenic bacterial strains provides the explanation of many phenomena and difficulties in studies of phage typing and specificity. A further effect of significance in this field is the possibility of obtaining hybrid viruses by genetic recombination between an externally infecting virus and a virus already symbiotically infecting a bacterial cell. These effects can be elicited experimentally but they have also been observed to occur spontaneously in stock cultures and, it is suggested, may account for processes of bacterial variation and evolution controlled by viruses.

SUMMARY: Further experimental evidence is described to support the concept that bacterial viruses exert a controlling effect on bacterial variation and evolution. A number of strains of diphtheria bacilli, both mitis and gravis, have been found to be carrying viruses capable of converting a susceptible avirulent diphtheria strain to full virulence and toxigenicity. The virus-resistant strains thus converted to virulence retain this property when subcultured and the change can be effected in vivo as well as in vitro. The mechanism of virulence transfer has been investigated, and it appears that this virus-controlled conversion to virulence cannot be explained on the basis of the selection of virulent mutants already present in the avirulent culture. Also Iysogenicity in a diphtheria strain does not itself imply virulence, since some viruses which attack the avirulent strain give rise to resistant cultures which are lysogenic and carry the infecting virus but they remain avirulent and non-toxigenic. It remains possible that the infecting virus transfers genetic properties such as virulence from the original virulent host bacterium to the infected strain. Selection by the infecting virus is also involved, and when a bacterial strain becomes infected with a mixture of viruses one of these becomes dominant in any definite cultural condition, infects all the cells in the culture and thus produces a ‘homogenizing’ effect. When precautions are taken to limit this spread of virus throughout the bacterial population it is found that ordinary laboratory cultures of ‘pure’ bacterial strains contain variant bacteria with distinctive properties. These when isolated are found to be infected with mutant viruses. The origin of the virus affects its virulence-conveying property; for example, virulence has not yet been transferred by a virus originating from an avirulent strain. On the other hand, some viruses carried by virulent diphtheria strains have been found to be incapable of conveying virulence, although they are able to render the avirulent culture lysogenic. Not only the origin but also the subsequent history of a virus affects its virulence-conveying power. One virus preparation was found to retain its virulence-conveying ability indefinitely when propagated on the avirulent strain, whilst after one passage through a susceptible virulent strain the ability was lost.

Interpretations of the phenomena associated with lysogenicity are complicated by the frequent occurrence of bacteria simultaneously infected with several distinct viruses and by the possibility of hybridization effected by genetic recombination.

SUMMARY: The treatment of bacteria with antibiotics, chemotherapeutic agents or various chemicals influences the nature of the viruses with which the bacteria are infected. In the ease of Corynebacterium diphtheriae many strains are infected with two or more distinct kinds of virus. Cultivation of these lysogenic strains in the presence of certain drugs may result in the removal of one of the viruses with a consequent alteration in the characters of the strain. Apart from changes in the host specificity of virus preparations yielded by the treated strains, these viruses may also differ in their capacity to transfer virulence to avirulent strains. For example, one diphtheria strain after treatment with certain antibiotics and chemicals developed the capacity to transfer virulence which it had not originally possessed. An avirulent strain after subcultivation in presence of certain drugs became susceptible to virulence-transfer from virus preparations not able to convey virulence to the parent strain. It appears that in lysogenic bacterial strains infected with two kinds of virus, one of the viruses may be more labile or easily removed than the other. Possibly the easily removable virus may be carried in the cytoplasm of the cell whilst the more stable virus may have some attachment to the nuclear apparatus of the bacterial cell. In one case investigated the stable virus was able to transfer virulence to an avirulent strain, whilst the cytoplasmic virus was not. It appears that there are two distinct mechanisms by which drugs may affect the viruses carried by bacterial strains: (i) by the selection of bacterial variants carrying a certain virus; (ii) by the preferential removal of the more labile viruses and inhibition of their multiplication. The results suggest criteria to be applied to the selection of potential chemotherapeutic agents for treating animal virus infections.

SUMMARY: Tetrahymena pyriformis S has an enzyme system which splits pyruvic oxime. This permitted the use of hydroxylamine as non-enzymic acyl acceptor in assaying pyruvate oxidation enzymes in crude extracts. Removal of thioctic acid from crude preparations of tetrahymena pyruvic oxidase by alumina treatment resulted in decreased acyl group formation, but did not affect the rate of oxidation in the presence of ferricyanide. Purification of the oxidase removed both the oxime-splitting system and the enzyme which is necessary for removal of thioctic acid from enzymes by the alumina procedure.

SUMMARY: Diaminopimelic acid decarboxylase was found to be widely distributed among 58 coli-aerogenes organisms. Different strains grown and tested under standard conditions showed great variations in enzyme activity although the amount of diaminopimelic acid in the cell hydrolysates was similar. Except for a higher average activity in Aerobacter aerogenes, there was no correlation between the activity of diaminopimelic acid decarboxylase and the biochemical type of coliform organism. No strain was found that contained diaminopimelic acid decarboxylase in the absence of lysine decarboxylase. The enzyme was also found in a strain of Pseudomonas aeruginosa but not in Proteus vulgaris, Streptococcus faecalis or Bacillus subtilis.