- Volume 40, Issue 1, 1965

Continuous culture and batch culture experiments on the control of morphogenesis of the aquatic phycomycete Blastocladiella emersonii led to the following conclusions: (1) The metabolism of Blastocladiella under the conditions of these experiments was highly oxidative, all of the substrate being accounted for as unused substrate, fungus-carbon and CO2. (2) Morphogenesis was dependent on potassium-ion concentration, ephemeral sporangiate organisms developed in low K+ concentration and resistant sporangiate organisms developed in higher K+ concentration. (3) Morphogenesis in these experiments was independent of CO2 partial pressure and pH value. (4) Morphogenesis was also dependent on an ionized species of carbonic acid (bicarbonate, carbonate) depending on the medium used. Ephemeral sporangia developed at low concentrations of these ions, K+, HCO3 – and CO3 2–, and resistant sporangia developed at higher concentrations. (5) The potassium and magnesium content and pH value of the organisms were independent of the pH value and potassium concentration of the medium over the ranges studied, and of the sporangium type produced by the organisms. The continuous culture apparatus used is described and continuous culture theory applicable to experiments in morphogenesis has been developed and tested.

The sequence of flagellar development accompanying differentiation during multiplication in a plate culture of Proteus mirabilis was investigated with the electron microscope and the negative-staining technique; the sequence of development can best be seen from the electron micrographs. The first flagella were produced towards the end of the first hour, and increased to a peak at about 6 hr and then decreased. The bacteria changed from coccoid to rod-shaped to elongated forms; the latter measured up to 80 μ, in length and were equipped with several thousand flagella. On the basis of measurements of flagellar complement, the elongated forms (or swarmers) can be regarded as ‘flagellin-factories’. The fine structure of both flagella and fimbriae was examined and several new features were seen, in particular certain structures at the bases of both appendages. The diameter of Proteus fimbriae was found to be about 40 Å.

Strains of Salmonella typhimurium and Escherichia coli which have acquired the colicine factor, colI, are less sensitive to the lethal effects of ultraviolet (u.v.) radiation than the non-colicinogenic parent strain. The dose of u.v. radiation required to kill 50% of the population of S. typhimurium strain LT2 colicinogenic for colicine I was greater by a factor of three than that required to kill 50% of the non-colicinogenic parent. The number of survivors of the non-colicinogenic strain decreased more or less exponentially with dose; the survival curve of the colicinogenic strain had a pronounced ‘shoulder’. Experiments with a strain of E. coli K 12 carrying lambda prophage indicated that the presence of colI decreased the incidence of phage induction following u.v. irradiation.

Phage lysins were prepared from high titre phage lysates of 3 strains of Streptococcus lactis, one of which was purified 500-fold by treatment with cold acetone, chromatography on Amberlite CG50 ion-exchange resin, and fractional precipitation from potassium phosphate buffers. The lysins were activated by monovalent cations and were similar in physical properties. Viable streptococci of groups N and D were lysed, but not those in groups A, B and C. Different strains of group N streptococci were lysed at different rates, the rate of lysis being characteristic for individual phage lysins. A lytic enzyme, prepared from purified phage particles by freezing and thawing or by ultrasonic treatment, showed the same pH optimum and specificity as the corresponding phage lysin. Streptococci were lysed-from-without in the presence of high multiplicities of phage. The type of lysin produced in phage-infected culture was genetically determined by the phage. Lysis of streptococci by phage lysin was inhibited by an unidentified substance present in crude phage lysates. The lysis of different strains of streptococci by particulate phage, phage-tail enzyme and phage lysin occurred at the same relative rates. This is interpreted as evidence for the localization of the phage-lysin substrate within the phage receptor site. Possible reasons for the different specificities of phage lysins from streptococci of groups N and C are discussed.

Cellular components in homogenates of brain tissue from scrapieaffected mice have been separated by centrifugation in sucrose and caesium chloride density gradients with the objective of location of the scrapie agent and concentration of scrapie activity. Improvements in the relative activity of the fractions removed from sucrose gradients were small but recovery was high in the material sedimented through 0·88 m-sucrose. No peak of activity was observed in zones removed from a caesium chloride gradient and activity throughout remained strongly associated with particulate matter. Ultrasonic disruption had little effect on scrapie activity. Concentrated preparations of sufficient potency for characterization of virus particles by electron microscopy were not obtained. From various experimental evidence the scrapie agent, if a virus, appears to be of small size. The strong association of the agent with tissue debris suggests that the presence of a tissue component may be necessary in the experimental transmission of the condition.

Preparations of the outer shell of Lactobacillus arabinosus were made by crushing the bacteria in a Hughes press and removing the cytoplasm by washing and centrifugation. Such preparations had none of the enzymes associated with glavin-mediated oxidation; they had consistently adenosine triphosphatase (ATPase) activity. The kinetics and other properties of this enzyme were studied. The evidence suggested that the enzyme was bound to the cytoplasmic membrane, the bulk of which appeared to be within the cell wall. The preparation was thus similar to that from obligate facultative aerobes and which, in contrast to the preparations from lactobacilli, contained enzymes associated with oxygen utilization mediated by the cytochrome hydrogen transport system. The bound ATPase had an optimum at pH 6·0 and was not markedly inhibited by ouabain or oligomycin: it was stimulated by 2,4-dinitrophenol. It was thus different from ATPase associated with ion transport but similar to that from mitochondrial membranes. There was no nucleotide pyrophosphorylase activity in the preparation but other nucleotide triphosphates and diphosphates and some inorganic phosphates were hydrolysed but at slower rates than was ATP.

A collection of tetrad-forming bacteria was divided into five groups by physiological and biochemical tests. Of these groups four were identified as species of the genus Pediococcus according to the classification of Nakagawa & Kitahara (1959). Useful differential tests were: pseudocatalase activity, catalase activity in the presence of heated blood, fermentation of glycerol and maltose, salt tolerance, pH sensitivity, final pH value in glucose broth culture, growth temperatures, arginine hydrolysis. The remaining group, composed of the type culture of Aerococcus viridans and a strain of Pediococcus urinae-equi, was identified as A. viridans. The pediococci were compared with enterococci and two species, P. pentosaceus and P. acidilactici, appeared to be physiologically and biochemically more like Streptococcus faecium than is S. faecalis.

At subinhibitory concentrations nystatin exerted its main effect on four species of Aspergillus in the lag phase of growth. Subsequent growth and sporulation appeared to be normal, indicating the lack of permanent damage to survivors. Starting with an inoculum of spores the increased lag period was caused by the inhibition of swelling and germination; starting with an inoculum of mycelium, by the temporary cessation of hyphal elongation. At low concentrations of nystatin the lag period increased in proportion to the increase in initial nystatin concentration. Under these conditions the length of the lag was limited by the natural breakdown of the antibiotic in the medium rather than by any metabolic activity or adaptive response of the fungi. However, the lag period increased sharply, as the inhibitory nystatin concentration for each species was approached, to values greater than can be explained solely by the increased concentration of antibiotic. This was not due to the time taken for growth by any especially resistant spores but to the greatly decreased viability and slow outgrowth of normal survivors. This loss in viability was due to factors other than K+ leakage. Plasmolysis of germinated spores and mycelium occurred in some Aspergillus species at high concentrations of nystatin. The action of nystatin was relatively slow; no significant loss of viability or lengthening of the lag phase was noted until after exposure to the antibiotic for at least one hour, even at very high concentrations. Since the fungicidal effect on Aspergillus terreus and A. fumigatus was appreciably slower than on A. niger and A. flavus, it is possible that differences in the rate of absorption of nystatin in part determine sensitivity to nystatin. Spore germination took place at higher nystatin concentrations than did hyphal growth. The lethal dose for spores, which was three to four times higher than the fungistatic value, was also higher than for the mycelium.

Sensitivity to ampicillin and cephalothin, the formation and induction of penicillinase and cephalosporinase, and the role of cephalosporinase in resistance to cephalothin were investigated in nine strains of Gramnegative bacteria. Naturally occurring strains resistant to cephalothin formed cephalosporinase, and ampicillin-resistant strains formed penicillinase. Cephalothin-resistant strains were all resistant to ampicillin, but the ampicillin-resistant strain of Proteus mirabilis was sensitive to cephalothin and did not form cephalosporinase. Penicillinase and cephalosporinase activity were induced by benzylpenicillin in five strains of Gram-negative bacteria and cephalosporinase was also induced by cephalothin and 6-amino-penicillanic acid in four of the inducible strains examined. Methicillin and quinacillin were variable in effect as inducers and cloxacillin partially inhibited cephalosporinase activity of two strains. Intrinsic resistance to cephalothin was high in four of five naturally occurring resistant strains.

Mutants of Saccharomyces cerevisiae impaired in the biosynthesis of carbamoyl phosphate were obtained. Genetical, physiological and enzymic studies of these mutants showed the existence in this organism of two independent enzymic systems which catalysed the synthesis of carbamoyl phosphate from HCO3 ‒, glutamine, ATP and Mg2+. One system provides carbamoyl phosphate for the arginine pathway, the other plays a similar role for the pyrimidine pathway. Carbamoyl phosphate from one pathway is freely available for the other. The mutations have been mapped in three unlinked loci. Two loci determine the argininespecific carbamoyl phosphate synthesizing system. The third locus corresponds to the pyrimidine-specific system. Mutations in either of the two genes concerned with the arginine pathway lead to a deficiency in the activity of that pathway to synthesize carbamoyl phosphate. Crossing a mutant deficient in one of the two arginine loci with a mutant deficient at the other produces a diploid in which complementation occurs. Also, in vitro activity may be partially regained by combining the cell-free extracts of the two single mutants. The physiological significance of the two enzymic systems is established by the study of their regulation. The carbamoyl phosphate synthesizing activity of the arginine pathway was repressed to 10% of its value in minimal medium by addition of excess of arginine to the growth medium. The activity corresponding to the pyrimidine pathway was only slightly repressed by uracil but was subject to feed-back inhibition by uridine-5′-triphosphate. These results are compared with the data available for other micro-organisms.

Identification is the practical application of taxonomic knowledge. Dichotomous keys and diagnostic tables form the backbone of everyday identification, but computers will be used in the future. Standardization of methods for characterizing tests, the development of multiple inoculation apparatus, and the use of mass cultures will enable more reliable tests to be carried out and more strains to be tested.