- Volume 107, Issue 2, 1978

Physiological tests were devised and used to compare 15 strains representing five species of soil and freshwater amoebae assigned to the genus Acanthamoeba. The tests gave an acceptable level of reliability and the pattern of responses was not affected by the differing growth rates of the organisms. There was some degree of overlap between the strains, as shown by the high level of inter-species similarities in relation to those between strains of the same species. Previous classifications of Acanthamoeba, which are based solely on morphological criteria, do not adequately reflect the diversity of these amoebae.

Heat-injured Streptococcus faecalis recovered tellurite resistance in 4 to 5 h when incubated at 33 °C in a non-growth medium containing KH2PO4/K2HPO4 buffer pH 7·1, glucose and casein hydrolysate. Only 10 % of the damaged population recovered in the absence of K+, or glucose, or casein hydrolysate, or in the presence of valinomycin in medium containing 1 mm-K+. The K+ content of cells increased two- to threefold on heating and a further three-to sixfold during the first 30 min of subsequent incubation. The Na+ content decreased simultaneously but Mg2+ concentrations were not affected. Recovery was not affected by adding 1 mm-EDTA or 2 mm-Mg2+ to the medium but the addition of 1 mm-EDTA and 2 mm-Mg2+ together reduced recovery to < 20 %. This effect was reversed by adding 2 mm-Ca2+.

A previous suggestion that sensitivity to ω-aminocarboxylic acids in Dictyostelium discoideum is associated with the ability to grow axenically has been re-evaluated using new axenic strains and axenic derivatives of strain ax3 recombinant for linkage group II. It is now shown that the major gene for ω-aminocarboxylic acid sensitivity, designated oaaA., is not allelic to previously described axenic loci axeA and axeB, but that oaaA is closely linked to, or allelic with a third axenic gene axeC found in strains ax2 and ax3. The axeC phenotype is rapid growth in axenic medium. The oaaA locus is located on linkage group II. Genetic evidence suggests that the strains ax2 and ax3 now in existence share a common genetic background and may not represent independent isolates from the wild-type strain nc4. Studies with 14C-labelled ϵ-aminocaproic acid show that sensitivity to ω-aminocarboxylic acids is correlated with increased myxamoebal permeability to these compounds.

Division of the nucleus and the cellular structure of bacteria may be correlated by using epifluorescent filter systems on acridine orange-stained material. The high resolution enables the spatial arrangement of double elements in the nucleus of Bacillus cereus to be visualized, after minimal handling, for comparison with information obtained by other methods.

Peptostreptococcus anaerobius VPI4330-1 is an anaerobic organism which has no superoxide dismutase, catalase or peroxidase. It can be protected from the toxic effects of oxygen by catalase in the culture medium. In order to elucidate its mechanisms of oxygen tolerance, the effect of oxygen on the metabolic activity of the organism was studied.

In salt solution supplemented with glucose or pyruvate the organism had a more rapid metabolic rate under aerobic conditions than under anaerobic conditions. There were also significant differences in metabolic end-products obtained under aerobic and anaerobic conditions. The crude cell-free extract had NADH oxidase activity, which reduced oxygen to water, and NADPH oxidase activity, which reduced oxygen to superoxide radicals and hydrogen peroxide. The former specific activity was much higher than the latter.

The results indicate that the main product of intracellular oxygen reactions was water. Deleterious products such as superoxide radicals and hydrogen peroxide were only formed to a limited extent. NADH oxidase may fulfil an important protective role in this organism.

Strains of Salmonella typhimurium representing various phage-types were permitted to swarm towards each other on soft agar to determine their compatibility. Some strains, regardless of phage-type, were totally compatible, whereas others regularly formed incompatibility zones. The widths of these zones varied from less than 1 mm to 12 mm. A detailed study of 30 strains of phage-types 1 and 42 showed that they belonged to a variety of incompatibility groups, while four strains of phage-type 179, which was only recently found in New Zealand, all belonged to one incompatibility group.

Observations by phase contrast, fluorescence and electron microscopy showed that epimastigotes of Trypanosoma (Schizotrypanum) dionisii (grown in vitro) were phagocytosed posterior end first by mouse peritoneal macrophages in vitro. Many were subsequently digested as a result of phagosome-lysosome fusion but others survived by apparently inhibiting this fusion and/or escaping from the phagosome into the host cell's cytoplasm. These survivors replicated as amastigotes. Long trypomastigotes, separated from populations grown in vitro by passage down a column of glass beads (with or without prior exposure to guinea-pig serum), were phagocytosed by either pole and all were subsequently digested.

Six different R-factors conferring trimethoprim resistance had been isolated from a variety of sources. The trimethoprim-resistant dihydrofolate reductases (EC 1.5.1.3) from strains containing these R-factors were purified by ammonium sulphate precipitation and DEAE-cellulose ion-exchange chromatography. The enzymes showed no significant differences in molecular weight, pH profile, substrate profile, heat sensitivity, inhibition profile and Michaelis–Menten kinetics. There was, however, considerable variation in the specific activity of these enzymes in the same bacterial host. When two Escherichia coli trimethoprimsensitive dihydrofolate reductases were examined as controls, considerable differences between their properties and those of the enzymes mediated by R-factors were detected. The data suggest that one trimethoprim resistance gene could be spreading through the bacterial population, possibly situated on a transposon.

At sub-bactericidal concentrations of hydrogen peroxide, Mycobacterium tuberculosis was killed by hydrogen peroxide/peroxidase/halide microbicidal systems. The halide cofactor could be either iodide or, with much lower efficiency, chloride. Omission of any one of the reactants eliminated the tuberculocidal effect. Differences in susceptibility between different strains of M. tuberculosis did not correlate with virulence differences. The observations are discussed in the context of host defence mechanisms against tuberculosis.

A structural study by methylation analysis was made of the lipopolysaccharides from Salmonella bredeney and strains of Salmonella typhimurium which had been lysogenized with phage P27 and thereby converted to have the new O-antigen factor 27. The results were compared with previous studies on the non-converted, 27-, parental strains. The change from 27- to 27+ was accompanied by a change in the linkage between the repeating units of the O side-chain from galactose-(1→2)-mannose to galactose-(1→6)-mannose. In S. bredeney (P27), where the expression of antigen 27 is stable, 98 % of the linkages were changed, whereas only 31 to 45 % of the linkages were affected in two strains of S. typhimurium (P27). As 71 to 85 % of the cells in the same batches of S. typhimurium bacteria were 27+ it seems probable that in these strains many bacteria contained both types of linkages in their lipopolysaccharide.

The properties of NADP+-linked glycerol dehydrogenase (EC 1.1.1.72) from Neurospora crassa were studied following 505-fold purification, with 54% yield, by gel filtration, ion exchange chromatography and affinity chromatography. Specific staining for the purified enzyme after disc gel electrophoresis revealed a single band and after isoelectric focusing, five bands. No enzymically inactive bands were detected by general protein staining in control gels. Total molecular weight was estimated to be about 160000, with a subunit molecular weight of about 43000. The forward reaction from glycerol to d-glyceraldehyde had a pH optimum of 9.5 and was specific for glycerol as substrate, since no activity was detected with other polyols tested. The reverse reaction had a pH optimum of 6.5 and was maximal with d-glyceraldehyde as substrate. K m values for glycerol and d-glyceraldehyde were 1.43 × 10–1 M and 1.15 × 10–2 M, respectively. Dihydroxyacetone also served as substrate in the reverse reaction. The enzyme was NADP+-specific.

Mutants (aspT) of Bacillus subtilis which lack the high affinity transport of l-aspartate and l-glutamate have been isolated by their resistance to dl-threo-β-hydroxyaspartate. They transport low concentrations (100 μ m) of all three amino acids at a greatly reduced rate but they can still grow at the normal rate on high concentrations of aspartate (25 mm) as sole carbon source. The aspT mutation has been mapped between argC and glpK, glpD. In glucose/citrate medium, an aspB mutant, auxotrophic for aspartate, requires much lower aspartate concentrations for growth than an aspB aspT double mutant. These results demonstrate that a low affinity aspartate transport system is still present in aspT mutant strains. This was also shown by the fact that both aspH and aspH aspT mutants, which carry the gene (aspH) for high aspartase production, grew at the same rate in media containing high concentrations (25 mm) of l-aspartate as sole carbon source. The high affinity aspartate transport system (K m = 67 μ mk) alone can satisfy the growth requirements of aspartate auxotrophs in media containing glucose or some other carbon source. However, the maximum rate (V max) of this transport system is too low to allow rapid growth on aspartate as sole carbon source. For such rapid uptake the low affinity aspartate transport system is needed. Membrane vesicles, energized by glycerophosphate, exhibit only the active high affinity transport which is saturated at about 100 μ m-l-aspartate (K m = 66 μ m). Apparently, the proton motive force, which energizes the high affinity aspartate (and glutamate) transport, is not (directly) required for the low affinity aspartate transport.

The blue–green alga Nostoc sp. strain Mac was grown heterotrophically in the dark on glucose and fructose. Cell composition was similar under photoautotrophic and hetero-trophic growth conditions and the efficiency of cell synthesis, in most cases, was similar to values obtained with other heterotrophic micro-organisms. Differences were seen in growth on the two sugars in response to sugar concentration and nitrogen source, and in the efficiency of cell synthesis in minimal medium, which indicated differences in the metabolism of the two sugars by the alga. Heterotrophic growth was stimulated by casein hydrolysate and dim light. Both effects were additive, indicating at least two different rate-limiting steps in the heterotrophic metabolism of the alga. Casein hydrolysate acted as a bulk nitrogen source for the alga and the response to dim light showed a chlorophyll a-like action spectrum.

The biosynthesis of a yellow–green, fluorescent, water-soluble pigment by Pseudomonas fluorescens occurred only when the bacteria were iron-deficient and was not directly influenced by the nature of the organic carbon source. The pigment formed a very stable Fe3+ complex and was purified in this form. Pseudomonas fluorescens produced only one molecular species of fluorescent pigment; however, its lability under mild alkaline conditions led to the formation of several pigmented decomposition products. The spectral properties of the pure pigment, its molecular weight (1500 ± 75) and its stability constant for Fe3+ (of the order of 1032) were determined. Both its biosynthesis and its chemical properties (formation of a stable Fe3+ complex) suggest that the fluorescent pigment is a desferrisiderophore.

Uptake experiments with 59Fe3+ showed that Pseudomonas fluorescens had an active system for iron transport. When the purified iron-binding pigment synthesized by this bacterium was added to the external medium, the rate of iron uptake by the cells increased significantly.

Addition of chemotactic attractant to Bacillus subtilis brought about a transient increase of absorption at 557 nm, compared with absorption at either 543 or 575·5 nm. The increase was tentatively attributed to reduction of cytochrome b. This reduction was linked to the ability of attractants (and certain other reagents) to make all bacteria in a population swim smoothly, rather than sometimes swimming and sometimes tumbling as they normally do. It is thought to signify a higher energy requirement for swimming since, in tumbling, flagella may tangle and jam, resulting in periods of no energy loss. Cations were required for motility; the function of the cations was probably not to energize motility, since protons alone could do that, but rather to reduce the surface potential of cells and, thus, avoid excess local acidity.

Arginine synthesis and urea formation via the ornithine cycle occurred in sporophore cap and stipe of Coprinus cinereus. Although urease was found at high activity in mycelium and stipe, it was not detectable in extracts of cap tissue, but arginine biosynthesis was specifically amplified during development of the cap as judged from metabolism of isotopically labelled substrates and increased enzyme activities. Four enzymes, NADP-linked glutamate dehydrogenase, glutamine synthetase, ornithine acetyltransferase and ornithine carbamoyl-transferase, were considerably derepressed in developing caps while remaining low (or declining) in activity in the stipes supporting those caps. Co-ordinate regulation of these enzymes could also be demonstrated in vegetative mycelium subjected to particular synthetic growth media. Arginine, alanine and glutamate accumulated in the cap as a result of amplified arginine biosynthesis. A greater than twofold increase in the quantity of urea in the cap was also evident during development, although the concentration of urea remained essentially unchanged. A causal relationship between urea accumulation and water influx into developing cap tissues is suggested. The inflation of the gill hymenium cells accounts for the ‘umbrella-like’ cap expansion that characterizes Coprinus spp.

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii incubated anaerobically in the presence of ampicillin utilized cellulose and to a lesser extent starch, but not soluble sugars. The protozoon incorporated 14C from 14C-labelled cellulose, glucose and starch; it synthesized protein from these compounds but only at a rate that would allow for the protozoa to divide every 8300, 1200 and 580 h, respectively, if this were the sole source of protein. These substrates were also metabolized with the production of acetic, propionic and butyric acids. Evidence is presented that 70 % of the cellulase inside the protozoa is soluble and not of bacterial origin. It had an activity of 11 to 28 pg cellulose digested h–1 protozoon–1 [0.55 to 3.3 μg cellulose digested h–1 (mg protein)–1].

The influence of amino acids on the synthesis of staphylococcal serine-proteinase (proteinase I) by Staphylococcus aureus strain v8 was studied. The repression of proteinase synthesis during the exponential growth phase was not due to any single amino acid in the medium. Proline was essential for proteinase synthesis in post-exponential growth but not for bacterial growth. At low concentrations of glycine, serine, threonine and proline, proteinase synthesis was inhibited, whereas at high concentrations of any of these amino acids, proteinase synthesis was fully induced. During post-exponential growth in the absence of glycine, serine, threonine or proline, the intracellular concentrations of alanine, aspartic acid and glutamic acid increased two to threefold. In the presence of these amino acids, the pool of alanine and aspartic acid remained at a low level and the concentration of glutamic acid decreased. The synthesis of proteinase I is probably repressed at high intracellular concentrations of glutamic acid.