- Volume 129, Issue 10, 1983

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT−. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli. strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.

Streptococcus mutans serotype c produces several extracellular proteins which bind to affinity columns of immobilized glucans. The proteins are three distinct glucosyltransferases and another glucan-binding protein (molecular weight 74000) which is now shown to be a fructosyltransferase. This enzyme is antigenically distinct and genetically independent of two other fructosyltransferases produced by the same organism. A mutant is described which lacks the glucan binding fructosyltransferase and has defective ability to form adherent colonies in the presence of sucrose. Although the production of glucans from sucrose results in the glucan binding protein becoming bound to the bacterial surface, and hence perhaps contributing to adherence, the fructans synthesized by the enzyme do not appear to contribute to this phenomenon.

Protoplasts were prepared from Streptococcus sanguis and some S. mutans serotypes by use of lysozyme (EC 3.2.1.17) under particular conditions: cells had to be grown in dl-threonine (20 mm) and harvested in early exponential phase. The efficiency of protoplast formation was enhanced by two additional steps: plasmolysis (in 12% PEG), prior to addition of lysozyme, and a swirling phase, after the enzymic action. This procedure allowed us to obtain clean protoplasts, with only 0·5% contamination by bacterial cell walls. Up to 90% protoplast lysis was obtained in 0·5 m-NaCl. Cytoplasmic membrane purification was achieved by centrifugation on a glycerol cushion.

The relationship between culture density or phase of growth at 24.5 °C and the ability of Candida albicans to form germ tubes when shifted to 37°C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.

Anoxygenic photosynthetic electron transport from sulphide, culminating in either H2 evolution or CO2 photoassimilation, was shown to include the segment from plastoquinone to ferredoxin in the cyanobacterium Oscillatoria limnetica. Both sulphide-dependent H2 evolution and CO2 photoassimilation were inhibited by plastoquinone analogues. In the former reaction, the block was bypassed by reduced N,N,N′,N′ -tetramethyl-p-phenylenediamine (TMPD). The link between this segment of electron transport and the hydrogenase enzyme was shown to limit the rate of sulphide-dependent H2 evolution. The rate of flow of electrons through this pathway was lower than would be expected either from the amounts of available enzyme, as measured by the in vitro oxidation of reduced methyl viologen, or from rates of electron transport to CO2 photoassimilation. When the strong reductant sodium dithionite was added to intact cells, the resulting low redox potential significantly improved photosynthetic sulphide-dependent H2 evolution. Hydrogenase activity in cell free extracts was similarly affected by dithionite. It is suggested that ambient redox potential controls electron flow through the hydrogenase, so that surplus reducing power is removed via H2 evolution.

The commonly used buffer HEPES was found to cause severe disintegration of the cyanobacterium Anabaena cylindrica, as measured by filament breakage, cell disruption, phycocyanin release and nitrogenase inhibition. The effect became increasingly severe as the buffer concentration was increased above 10 mm. The observed cell damage does not appear to be unique to HEPES, similar observations being made with Tris/HCl, sodium phosphate, sodium sulphate and sodium chloride. It appears that the cells are very sensitive to ionic strength.

Cultures of the Mollicutes (mycoplasmas) Acholeplasma laidlawii B, Acholeplasma morum, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma fermentans and Mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in pH and glucose concentration, and concentrations of ATP, ADP, AMP, lactate and pyruvate. The adenylate energy charge (ECA), the mean generation time, and the number of nmol of ATP (mg dry weight)−1 were calculated for cultures in the mid-exponential growth phase. The maximum cell concentrations ranged from 0·2 1010 to 5·0 1010 c.f.u. ml−1. Doubling times ranged from 0·34 to 3·29 h. The fermentative, non-arginine-requiring A. laidlawii B, A. morum, and M. gallisepticum, as well as the fermentative, arginine-requiring M. fermentans, utilized glucose and produced lactate and pyruvate. The non-fermentative, non-arginine-requiring M. bovis neither utilized glucose nor produced lactate or pyruvate. The non-fermentative, arginine-requiring M. arginini utilized glucose, but did not produce lactate or pyruvate. At mid-exponential growth phase, the average ECA of A. laidlawii B was 0·90, a value similar to that reported for Spiroplasma citri and other bacteria. In contrast, the average ECA of A. morum and the four Mycoplasma species was 0·70. In A. laidlawii B at mid-exponential growth phase, ATP accounted for 97% of the total adenylate nucleotide pool. At the same stage of growth, the average cellular ATP concentration of the other Mollicutes was significantly lower, ranging from 45 to 63% (P < 0·01). Excluding A. laidlawii B, the Mollicutes were relatively energy deficient during their mid-exponential growth phase. The diminished metabolic capacity may be related to the association of Mollicutes with living cells and perhaps to the cytopathic effects of these micro-organisms.

Two methylotrophic bacteria having similar cell morphologies have been isolated from cultures of ‘Methylobacterium ethanolicum’ grown on methane. One of these, strain POC, is an obligate methanotroph containing the serine pathway and Type II intracytoplasmic membranes, which appears to be a ‘Methylocystis’ species. The second organism, strain H4-14, can fix N2 and grows on a variety of substrates, including methanol, formate, ethanol, succinate, fructose and H2 + CO2; during growth on methanol, carbon is assimilated via the Calvin-Benson cycle. Strain H4-14 appears to be a Xanthobacter species. Mixtures of the two organisms formed stable associations during growth on methane, consisting of approximately 5–20% H4-14 and 80–95% POC. Strain POC excreted biotin, which was required by strain H4-14. Our results suggest that ‘Methylobacterium ethanolicum’ cultures grown on methane consist of a syntrophic association of two methylotrophs.

The initial metabolism of some methylated and ethylated amines, which were used as a nitrogen source but not as the sole carbon source by the yeasts Candida utilis and Hansenula polymorpha, involved a peroxisomal amine oxidase which produced ammonium ions, hydrogen peroxide, and formaldehyde or acetaldehyde. The aldehydes so formed were either oxidized via their corresponding carboxylic acids or, depending on the organism and the aldehyde, also partly assimilated into cell material. The synthesis of amine oxidase, which was paralleled by the development of peroxisomes in the cells, was repressed in the presence of ammonium ions and derepressed under nitrogen limitation. Amines were not required as inducers of enzyme synthesis. Utilization of ethylated amines, but not of methylated amines, as a nitrogen source resulted in a significant increase in cell yield. In both yeasts ammonium ions were assimilated mainly by way of NADPH-dependent glutamate dehydrogenase. The activity of this enzyme increased drastically in cells grown under ammonium or amine limitation or, under carbon limitation, in the presence of amines as the sole source of nitrogen. Under the latter conditions free ammonium was not detectable in the culture supernatant, while the amount of amines utilized was just sufficient to account for the amount of cell material produced. This indicated that during growth with amines as the nitrogen source the physiological condition that the cells experience is in fact one of ammonium limitation. Our results suggested that the rate of amine oxidation was determined by the intracellular concentration of ammonium and determined via repression of amine oxidase synthesis. Due to this control and the high nitrogen to carbon ratio of amines, sustained growth of the methylotrophic yeast H. polymorpha on methykmine and of both species on ethylamine as a carbon source is not possible, even though these organisms are able to grow on the related compounds methanol and ethanol.

Flagella from Bacillus firmus RAB, an alkalophilic bacterium, were purified to homogeneity. The flagella were shown to consist of a single protein subunit (flagellin) with an apparent molecular weight of 40000. The amino acid composition of B. firmus RAB flagellin was similar to that of other bacilli except that the former had far fewer basic amino acids. The paucity of basic amino acics may render the flagella more stable at external pH values as high as 11·0.

Two new ballistosporogenous yeasts, Bullera crocea and Bullera armeniaca, are described. Bullera crocea was isolated from strawberries, cauliflowers and watercress, and B. armeniaca from cauliflowers and cabbage. Unlike B. alba, B. dendrophila, B. piricola and B. tsugae, these new species could not utilize lactose; B. crocea was distinguishable from B. armeniaca by the ability of the former to utilize melibiose.

Ureaplasmas isolated from sheep and goats were compared by immunofluorescence with antisera prepared in calves and by PAGE of polypeptides labelled by growth in the presence of [35S]methionine. The ovine and caprine strains constituted two groups defined by serology and polypeptide composition that were not related to the animal species from which they originated. Strains representing these two groups were compared with Ureaplasma urealyticum (human isolates) and U. diversum (bovine isolates). They could not be classified with either but were more similar to the U. diversum strains.

Extracellular glycosyltransferases from 17 strains of Streptococcus mutans were examined by analytical isoelectric focusing. Three kinds of glucosyltransferase: highly-branched-1,6-α-d-glucan synthetase, 1,3-α-d-glucan synthetase and 1,6-α-d-glucan synthetase, were excreted from serotype a, d and g strains. The enzymes of serotype a strains were distinguishable from those of serotypes d and g by differences in their pI values. Serotype c, e and f strains excreted basic glucosyltransferase and acidic fructosyltransferase. Serotype b strains also excreted the glucosyl-and fructosyltransferases, but the pI values were different from those of the enzymes from the other serotypes. Thus, S. mutans strains could be divided into four groups by analytical isoelectric focusing of glycosyltransferases which corresponded well to the four genetic groups.