- Volume 24, Issue 1, 1961

Relative differences in radiosensitivity between Escherichia coli strain B and the mutant strains, B/r (more resistant than B) and two types of Bs (more sensitive), are maintained with respect to inactivation by ultraviolet radiation, X-rays and decay of incorporated 32P. The differences cannot be ascribed to variations in number of nuclei or in content of DNA or RNA. The effectiveness of various postirradiation and post-decay treatments in preventing inactivation varies for each strain. With the exception of reactivation by light, strain B/r is less reactivable than B and Bs.

The strains do not differ in their rates of mutation to resistance to bacteriophage T1. However, the rates of mutation of the Bs mutants to the first step of furacin- resistance are considerably lower than the rate for the parent strain B. From one of the Bs mutants, new strains were obtained whose properties are mixtures of those of B and B/r.

A procedure is described for measuring the viabilities (% viable/total organisms) of populations of Aerobacteraerogenes by short-term incubation on agar films followed by differential counting under the microscope. The only special apparatus needed is a supply of metal annuli. The procedure estimates viability with 95 % fiducial limits of ± 9 %; it appears applicable to other aerobic bacteria, to yeasts and to spore suspensions.

A Pediococcus sp., unusual in that it needed reducing conditions for growth, would not grow in a medium based on acid-hydrolysed casein but grew when asparagine was added. Neither glutamine nor aspartic acid + an ammonium salt satisfied this requirement. When the acid-hydrolysed casein was replaced by a mixture of amino acids, the omission of asparagine caused a decrease in growth in the presence of aspartic acid + an ammonium salt. No growth occurred in the absence of both aspartic acid and asparagine.

The oxidation of 20 carbohydrates and derivatives by 45 strains of acetic acid bacteria, representing most of the known species, was studied and correlated with the enzymic constitution of the organisms. The strains of the mesoxydans group of Frateur and the acetate-oxidizing Gluconobacters oxidized the greatest variety of substrates and contained the most complex enzyme system. The strains investigated could be arranged in two main lines, each one with a stepwise decreasing gradation of oxidative properties. In contrast to the expectations evoked by their names, the strains of the ‘oxydans’ group had only limited oxidative powers and the ‘per- oxydans ’ bacteria even less so. The enzymic mechanism of the catabolism of several carbohydrates was discussed. It is proposed to split the acetic acid bacteria into two biotypes: Gluconobacter oxydans and Acetobacter aceti, and to consider the existing species as varieties within the two main types. An approach to the phylogenetic interpretation of the intermediary carbohydrate catabolism of these bacteria is discussed.

Glycolysis of yeast is inhibited by the polyene antibiotic nystatin. This inhibition was prevented or reversed at or above pH 5·8 (the approximate intracellular pH of yeast) by adding NH+ 4, K+ or Rb+ (in order of decreasing effectiveness). These ions do not interfere with the absorption of nystatin by the yeast cell. At pH 4 both glycolysis and respiration are highly sensitive to nystatin but are not protected by the monovalent cations. At pH 7 respiration of intact cells was insensitive; only with yeast protoplasts could respiration be inhibited by nystatin. Temporary protection was obtained with NH+ 4or K+.

K+ and NH+ 4did not prevent the fungicidal or fungistatic actions of nystatin even under conditions where the protection of glycolysis by these ions was essentially complete. The original yeast cells were not dependent upon added K+. After a brief treatment with nystatin at pH 7·0, the cells showed an absolute requirement for K+ or NH+ 4; it was shown that K+ was rapidly lost by cells following contact with nystatin. It is concluded that nystatin directly damages the cell membrane and thus produces a rapid increase in permeability to small ions. The resulting depletion of cellular K+ halts glycolysis. The addition of K+ or NH+ 4 restores glycolysis but does not reverse the membrane alterations.

Phage-immune variants were detected among chromogenic acid-fast mycobacteria by the action of Mycobacterium phlei bacteriophage. One of the strains thus obtained showed not only phage immunity, but also a marked difference in colonial appearance, pigmentation, antigenic composition and pathogenicity for laboratory animals as compared with the original strain.

A strain of Saccharomyces cerevisiae, grown in a medium containing a suboptimal concentration (0·4 × 10−10m) of biotin, was shown to contain less deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein but, during the early stages of growth, increased concentrations of acid-soluble ultraviolet (u.v.)-absorbing substances, as compared with the same organism grown in the presence of an optimal concentration (8·0 × 10−10 m) of biotin. The concentration of acid-soluble u.v.-absorbing substances in the biotin-deficient yeast was higher, irrespective of the nature of the extracting acid (0·2v-perchloric acid, 5% (w/v) and 10% (w/v) trichloroacetic acid, or 5 % (v/v) n-butanol in m/15 KH2PO4). Raising the temperature of extraction from 3° to 21° or 30° had little or no effect on the amounts of these u.v.-absorbing substances extracted. Analyses of the nucleotides and nucleo- bases in the yeast RNA showed these to have a ratio of purine:pyrimidine bases of 1·00–1·15, with the exception of the RNA from 5-day cultures of biotin-deficient yeast which had a slightly but consistently higher ratio. The significance of these results is discussed in relation to the metabolic function of biotin.

Media have been devised containing tracheal hydrolyzates or d-galactose as an economical means for the cultivation of large quantities of galactosamine-meta- bolizing bacteria. With these media Lactobacillus bulgaricus and L. casei showed good growth; dried preparations were able to utilize galactosamine to produce ammonia and lactic acid.

With synchronous single-generation cultures of the non-filamentous water fungus Blastocladiella emersonii the activities of isocitric dehydrogenase, α-ketoglutaric dehydrogenase, and cytochrome oxidase were studied during morphogenesis. The total isocitric dehydrogenase activity per plant increased rapidly following germination of the spore and reached its peak at the point of no return in ontogeny; this increase was about seven times greater than the increase in total α-ketoglutaric dehydrogenase activity. During growth from the spore stage to the stage of irreversibility, the specific activity of isocitric dehydrogenase increased 350% while that of α-ketoglutaric dehydrogenase decreased 50%. By removing the external bicarbonate before the point of no return was reached, and thus reversing the morphogenetic pathway, the specific activity of isocitric dehydrogenase, which was in the process of increasing, decreased immediately, while the α-ketoglutaric dehydrogenase activity, which had decreased sharply, increased quickly once again. Similarly, the total activity of isocitric dehydrogenase per plant decreased precipitously while, without reversal, it continued to rise. Conversely, reversal of the morphogenetic path caused an immediate rise in the total α-ketoglutaric dehydrogenase activity per plant while, in the absence of reversal, enzyme activity remained on a level plateau. Thus, removal of bicarbonate induced a new morphogenetic path and, simultaneously, a 250% increase in the activity of one key enzyme and a 35 % decrease in the activity of a second; on the other hand, the total protein and total activity of two other ‘control’ enzymes (glucose-6-phosphate dehydrogenase and cytochrome oxidase) remained essentially constant. In contrast to the above, removal of bicarbonate immediately after the point of no return in morphogenesis had no significant effect on these same enzymes. The data provide substantial evidence that a reversible bicarbonate-induced enzyme synthesis is involved in morphogenesis; i.e. in the mechanism of the bicarbonate-induced formation of resistant-sporangial plants in Blastocladiella.

The endogenous respiratory rate of non-photosynthetic Euglena gracilis var. bacillaris is remarkably constant under varied conditions of growth and incubation. After 60–90 min. of slightly more rapid respiration, the endogenous oxygen uptake averages about 6·5 μl. O2/million cells/ hr., and this rate can be maintained for at least 26 hr. The respiratory quotient of the endogenous metabolism is 1·0. Fluoroacetate inhibits endogenous oxygen consumption about 70 %. A variety of evidence suggests that the polysaccharide paramylum is the major endogenous substrate.

Tracer experiments indicate that the endogenous metabolism continues during the oxidation of exogenous substrates, but that the assimilated substrate mixes with, and dilutes the endogenous reserves. The time-course of this dilution process indicates that the most recently assimilated reserves are the first to be oxidized.

SUMMARY: The fixation of tetanus toxin by brain ganglioside has been confirmed. A method has been developed for assaying the toxin-fixing capacity of ganglioside in the analytical ultracentrifuge. Under appropriate conditions ganglioside will fix many times its own weight of toxin. Ganglioside preparations from brain contain at least three gangliosides; at least two of these differ in their sialic acid contents and toxinfixing capacities. The sialic acid residues (and their free carboxyl groups) are essential for toxin-fixation. Tetanus toxin does not appear (so far) to bring about any change in the ganglioside molecule.

SUMMARY: The capacity of a number of gangliosides to fix tetanus toxin and to inhibit haemag- glutination by influenza virus was investigated. Toxin fixation increased with total sialic acid content but not in strict proportion. Whether or not the sialic acid is bound by neuraminidase-labile linkage appears to be irrelevant in determining whether toxin is fixed. Viral inhibition by ganglioside also is related to the total content of sialic acid; but there exists at least one ganglioside which has very little capacity to react with virus and whosesialic acid is virtually all insensitive to neuraminidase. Tetanus toxin does not prevent neuraminidase from attacking ganglioside.

SUMMARY: The observation that growth in the presence of methionine results in organisms with a greatly decreased ability to form methionine from homocysteine has been extended to other strains of Escherichia coli, in particular strain PA 15, an organism for which there is a considerable background of knowledge concerning the enzymic mechanism of this stage of methionine synthesis. When organisms grown in the absence of methionine (‘ active ’ organisms) are transferred to a medium containing methionine the activity is not lost, but simply diluted by new organisms having much less activity. Both organisms and ultrasonic extracts derived from them show the same comparative loss of activity when the organisms are incubated with methionine.

Organisms grown in the presence of methionine (‘inactive’ organisms) regain activity when incubated in a growth medium without methionine. The major part of such regeneration of activity occurs before growth is significant. Enzyme formation occurs linearly and without lag and is stopped by the addition of methionine at any stage. A number of analogues of methionine also repress enzyme formation, but none is as active as methionine.

Regeneration of enzyme is inhibited by chloramphenicol and is greatly diminished when the supply of individual amino acids is restricted either by the use of an inhibitory analogue (p-fluorophenylalanine) or by the use of amino acid-requiring auxotrophs. Formation of enzyme therefore requires de novo synthesis of protein. Similar techniques gave no evidence that concurrent synthesis of ribonucleic acid was also required.

Compounds which act as substrates or cofactor for methionine synthesis (serine, homocysteine, cobalamin) also repressed enzyme formation to some extent and, after a lag, full repression was obtained with a mixture of all three. These effects are probably due to methionine itself whose synthesis by the enzyme initially present is stimulated by the test substances.

SUMMARY: Members of the genus Aeromonas produced extracellular toxic antigens which were specific for this group and antisera produced against these antigens could be used to identify these organisms. Strains of A. liquefaciens, A. punctata, A. hydrophila and A. formicans were apparently very closely related; differences which existed between them were not greater than differences found among the strains of A. liquefaciens. Fresh isolates tended to be strongly haemolytic, produced extracellular toxins which were lethal to mice and elicited haemorrhagic lesions upon injection into the skin of rabbit. Old laboratory cultures tended to be much less haemolytic and less toxigenic. A. salmonicida appeared to possess extracellular antigens specific for this species but some strains of A. liquefaciens were found to possess this antigen.

Members of the genus Serratia were included in this study since Stevenson (1959) suggested that all Aeromonas strains are non-pigmented forms of Serratia. All strains of Serratia examined, regardless of their classification into species, were homogeneous and reacted with antiserum produced with one strain. Moreover, no cross-reaction was found between the antigens of Aeromonas and Serratia and it appeared unlikely that Aeromonas could be regarded as a non-pigmented member of Serratia. No cross-reaction was found between the extracellular antigens of these two genera and the other members of the families Enterobacteriaceae and Pseudo- monadaceae and antisera produced with these antigens appeared useful in the identification of Aeromonas and Serratia.

SUMMARY: The inhibitory effect of low temperature (c. 10°) on initiation and early development of zygospores of Rhizopus sexualis is less severe in mature cultures (in which numerous zygospores have already been formed before transfer to the low temperature) than in young ones. The severe effect of low temperature on immature cultures is not counteracted by any of a number of known growth substances, by extracts of mature mycelium and zygospores, or by used culture media. The effect of mature cultures in counteracting low temperature inhibition of zygospore production in young ones is able to pass across a gap of 5 mm. between the cultures and must therefore be due to a volatile substance (or substances) produced by the mature mycelium. By the use of small chambers which allow young test cultures to be kept at 10° while a stream of air from mature ones growing at 20° passes over them, it is confirmed that the active factor is volatile. This substance is not carbon dioxide or ammonia and is basic in nature. Its probable identity is discussed and comparison is made with some other volatile substances reported to influence growth and development of fungi.