- Volume 50, Issue 2, 1968

Mutants of Pseudomonas aeruginosa 8602 were isolated which were unable to grow with acetamide as sole carbon source. They were divided into five classes on the basis of growth studies and enzyme assays. The mutants able to grow on acetate but not acetamide (Am–) lacked an inducible amidase. Mutants unable to utilize either acetamide or acetate (At–) lacked one of the glyoxylate cycle enzymes (isocitrate lyase or malate synthase) or were deficient in citrate synthase or acetic thiokinase. Isocitrate lyase-negative mutants grew on propionate which is therefore not metabolized by the glyoxylate cycle. The amidase marker was not co-transduced with the isocitrate lyase, acetic thiokinase or citrate synthase markers.

The growth requirements of a vitamin B12-dependent soil bacterium (strain 544), provisionally classified as belonging to the genus Arthrobacter, were studied. The organism showed a high specificity for vitamin B12; of the naturally occurring analogues tested, only α-(5-hydroxybenzimidazolyl)-cobamide cyanide and α-(2-methylmercaptoadenyl)cobamide cyanide possessed some growth-promoting activity (less than 10%). The anilide and the ethylamide of the monocarboxylic acid of vitamin B12 inhibited growth and the inhibition was reversed by the vitamin. A medium and a procedure for vitamin B12 assay using strain 544, is described. Comparative assays with Ochromonas malhamensis and Escherichia coli 7m are given and their differential usefulness for differentiating the various congeners of vitamin B12 discussed.

Addition of vitamin B12 to B12-deficient cell suspensions of strain 544 stimulated the biosynthesis of methionine and the oxidation of propionate, valerate, isoleucine and valine. The endogenous respiration and oxidation of glucose, butyrate, leucine and lysine were only slightly enhanced by vitamin B12.

A small amoeboid organism, found in mammalian tissue cultures inoculated with an infective agent formerly termed ‘Ryan virus’, is shown to have the morphological, cultural and behavioural characters of the free-living soil amoeba Hartmannella castellanii Douglas, 1930. Cytopathic changes occurred regularly in the infected monolayers; this was evidently due to action of the amoebae rather than the presence of any associated bacterial or viral agents. Strong circumstantial evidence suggests that the Ryan isolates of H. castellanii originated, either as trophozoites or cysts, from swabs of the human nasopharynx. Recovery of hartmannellid amoebae from this source is of interest in relation to some recently reported cases of pyogenic meningitis, apparently caused by free-living soil amoebae.

Myxamoebae of Physarum flavicomum and the stages of their development of flagella during transformation into biflagellate swarm cells were examined with the electron microscope. Fixation was with glutaraldehyde osmium. The flagellation process was similar to that reported in other systems, involving budding of the basal body into a primary flagellar vesicle which then forms the flagella sheath. Two basal bodies are present in the myxamoeba prior to flagellum development; other morphological features of myxamoebae and swarm cells, including systems of microtubules, are described. Mitochondria of both stages contain a dense core, flagella exhibit the typical ‘9 plus 2’ arrangement, and the doublet fibrils of the axoneme become singlets near the rounded distal end of the flagellum.

Forty-six of the 48 strains named Streptomyces griseus in our collection have in common a distinctive pattern of properties and are believed to represent a recognizable taxonomic group, or aggregate, of this polytypic species. Seventy-seven additional strains bearing 51 different species names were found to possess the same pattern of characteristics. The distinguishing properties of this aggregate are described and compared with those of S. albus, S. fradiae and S. somaliensis.

Twenty-six strains of Nocardia dassonvillei (Brocq-Rousseu) Liégard &Landrieu from the soil and from infections of man and animals are described, and a pattern of characteristics for their identification and differentiation from N. asteroides, N. brasiliensis, N. caviae, N. madurae and N. pelletieri is presented. Strains of N. dassonvillei resembled strains of Streptomyces griseus (Krainsky) Waksman &Henrici in the macroscopic appearance of their growth and in many of the physiological properties examined.

A fertile mutant (fer) isolated from Salmonella typhimurium lt-7mut with a mutator gene mut was found to have a high recipient ability when used as a recipient of the Escherichia coli chromosome and F′ and R factors, whereas lt-7mut + and S. typhimurium lt-2mut +, both of which lack the mut gene, were poor recipients of these foreign deoxyribonucleic acids, lt-7mut exhibited an intermediate recipient ability. These episomal elements, however, were transferred from lt-7mut and lt-7mut + to the mut, mut + and fer substrains at frequencies comparable to an E. coli recipient. In contrast, the frequencies of transfer of these episomes from lt-7fer to the other substrains of S. typhimurium were considerably lower than those to lt-7fer. The efficiencies of plating (e.o.p.) of phage P-22 grown on lt-7fer were likewise lower on lt-7mut, lt-7mur + and lt-2mut + than on lt-7fer. The e.o.p. of phage P-22 on lt-7mut, lt-7mut + and lt-7mut + were increased to the value on lt-7fer by its growth on lt-7mut + or lt-2mut +. The frequencies of transduction of an R factor to these substrains of S. typhimurium with phage P-22 grown on various substrains were parallel with the e.o.p. of this phage on these strains. It was further shown that the deoxyribonucleic acid of phage P-22, which had previously been propagated on lt-7fer, injected into lt-7mut + and lt-2mut + is rapidly degraded, whereas appreciable breakdown did not occur in lt-7fer. These results with P-22 indicate that lt-7fer is a mutant which is impaired in its capacity of restriction and modification of this phage. The growth of P-22 on lt-7mut resulted in partial increase of its e.o.p. on lt-7mut + and lt-2mut +, indicating that the restriction and modification capacities of lt-7mut are partially affected. The above results, that lt-7fer acts as a good recipient in the conjugal transfer of E. coli chromosome and non-viral episomes and that the frequencies of transfer of the non-viral episomes among the substrains of S. typhimurium are comparable to those to lt-7fer and E. coli, are interpreted as due to the restriction- and modification-less nature of lt-7fer.

Micro-organisms in aqueous suspensions were killed when voltages above a certain threshold value were discharged through electrodes immersed in the suspensions. The relation of the peak pressure, peak current and the arcing time to the killing effect were studied, but no clear-cut relationship was ascertained. At the discharges shock waves with durations of 20–105 μsec. and pressure amplitudes of 40–250 bar were generated. Although bigger and more solid objects were considerably damaged, the micro-organisms were inconspicuously affected by the pressure shock wave alone.

The importance of photon radiation for the microbicidal effect of electrical discharges in water has appeared from a series of experiments. (a) An ultraviolet (u.v.)-sensitive strain of Escherichia coli was more susceptible to discharges than a u.v.-resistant mutant derived from it. (b) The kinetics of the inactivation by discharges were under certain conditions similar to those of continuous u.v. irradiation. (c) Addition of u.v.-absorbing substances to the discharge liquid decreased the bactericidal efficiency of the discharges to an extent which paralleled the u.v. absorbance. (d) The bactericidal effect decreased as the distance from the spark increased. (e) Bacteria enclosed in a cellophan bag were killed by discharges outside the bag, even when the bag was kept in the air above the discharge liquid. (f) Also discharges in air were active. (g) Bacteria inactivated by discharges were accessible to photoreactivation, but the magnitude of the reactivation was generally less than that obtained with bacteria inactivated by continuous u.v.-irradiation. In addition to direct radiation effects other kinds of microbicidal activities were produced by the electric discharges.

Hypocholesteremic compounds, triparanol, SKF 525-A, SKF 3301-A, SKF 16467-A, inhibited the multiplication of some bacteria and ascomycetes; phycomycetes were not inhibited. Gram-positive bacteria were most sensitive to these compounds. Dimethyl or diethyl aminoethanol and SKF 2314, compounds resembling the major moieties of the SKF compounds, were not inhibitory alone or together; the whole molecule was necessary for inhibition. Inhibition by triparanol of the multiplication of several Gram-positive bacteria was annulled by albumin and ergosterol; oleic acid and squalene were mostly inactive.

A small number of organisms in cultures of the dinoflagellate Gonyaulax monilata attained twice the diameter of normal organisms and showed an excessive accumulation of carbohydrate reserves. The nuclei of these giants degenerated as did the cytoplasm. It is suggested that this was not a response to cultural conditions but represented an altered metabolism of individual organisms.

The effects of two drugs (chloramphenicol, actinomycin D) which inhibit protein synthesis on the nucleus of the dinoflagellate Gonyaulax monilata were examined. Chloramphenicol had no effect on the morphology of the nucleus but caused a diminution of cytoplasmic protein and a suppression of growth; did not completely suppress mitosis. Treatment with actinomycin D resulted in the loss of chromosomal arrangement within the nucleus, the fraying of the edges of the nucleus and the loss of the nuclear ‘band’, and suppressed division. The results are thought to support the hypotheses that the matrix functions in maintaining nuclear morphology and that the ‘band’ is necessary for mitosis.

The rates of growth of five strains of Aspergillus niger (m, mubli, nrrl 334, nrrl 323, nrrl 346) were followed in an optimum nutrient solution. The time course of growth of all the strains showed a steep decrease after 3 days, except that of strain NRRL 346 which tended to flatten out. A correlation between sporulation and mycelial dry weight was observed. All the five strains showed a marked decrease in mycelial yield in the absence of manganese from the basal medium. Their quantitative responses to graded concentrations of Mn in the medium were compared with spore and mycelial inocula. Statistical analyses of the result suggest that strain mubli is the most suitable test organism for the bioassay of Mn. The effective range of concentrations of Mn which coincided with the maximum differences in mycelial dry weights was 0·001–0·006 μg./ml.

Two aerobic mesophilic microbial species of a new genus belonging to the family Streptomycetaceae of the order Actinomycetales are described under the name Microtetraspora (Microtetraspora glauca, type species). The microorganisms produced a filamentous growth which is differentiated into a vegetative and an aerial mycelium.

The new genus is characterized by the formation of a short and sparsely branched aerial mycelium bearing at the end of short sporophores chains of four spores. Sporulation is not observed to take place on the vegetative mycelium. The genus Microtetraspora is widely distributed in the soil and has been isolated from many samples.

The base compositions of the deoxyribonucleic acid (DNA) and ribo-nucleic acid (RNA) of three strains of Mycoplasma laidlawii and one of Mycoplasma mycoides var. mycoides have been determined. The adenine (A) + thymine (T) content of all the samples examined was much greater than the guanine (G) + cytosine (C) content, the values for % GC ranging from 34·4 to 32·5 for the sample of M. laidlawii to 30·0 for M. mycoides var. mycoides. The adenine + uracil/guanine + cytosine value for the RNA of all the samples was in the range 1·17–1·20.

The amino acid content of the protein of Mycoplasma laidlawii was also determined. This agrees closely with the values obtained by Sueoka (1961) for the amino acid composition of the protein for a bacterium having the same DNA base composition.

The values given in a previous communication (Jones, Tittensor & Walker, 1965) for the amino acid composition of the protein of Mycoplasma mycoides var. capri were incorrect since a protein fraction had precipitated from the medium during the growth of the organism because of the decrease in pH value of the culture which occurred. The correct values are now given and they agree closely with the values predicted from the work of Sueoka (1961).

Thirty-eight strains of Corynebacterium acnes were used in a systematic study of biochemical and physiological reactions in an effort to define and clarify some of the characters of this species. Strains were isolated both from lesions in acne vulgaris and from normal skin.

The characters which were positive for all 38 strains were: gelatin liquefaction within 20 days, catalase production, ammonia production in peptone broth, tributyin hydrolyis, α-haemolysis (green) in blood agar containing human or rabbit red blood cells, acid production from fructose, glucose, galactose, mannose, glycerol and trehalose.

The experimental pathogenesis of Aeromonas salmonicida for the sea trout (Salmo trutta) and the brown trout (Salmo fario) in sea and brackish waters was examined by contact between infected and healthy fishes. Sea trout and brown trout both acquired infection by contact with infected S. fario at salinity values from 2·54% to 3·31% (w/w) when water temperatures ranged from 5·6° to 14·5°. The species differed in their resistance to infection. About 90% of the sea trout became infected and died within 7 days, developing superficial lesions typical of the disease. The brown trout appeared to be more resistant, about 75% developing and dying from an atypical form of the disease.

Thirty-six strains of enterococci were examined by standard physiological tests. The soluble proteins of cell-free extracts were separated by electrophoresis in starch gels. Specific staining technique for glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were applied to the gels. The results of electrophoresis supported the subdivision of the group into two major divisions represented by Streptococcus faecalis and S. faecium, with S. durans as a variant of S. faecium. It was possible tentatively to identify three strains which did not fit well into the taxonomic scheme used.