- Volume 89, Issue 1, 1975

SUMMARY: Exopolysaccharides were prepared from cultures of four Myxococcus strains grown on solid and in liquid media, and also from the fruiting bodies. Lipopolysaccharides could be extracted with aqueous phenol from the vegetative bacteria, but were absent from microcysts. Mannose and d-glucose were present in all the exopolysaccharides and three of the lipopolysaccharides examined. Other monosaccharides identified in the exopolysaccharides were d-galactose, N-acetylglucosamine and N-acetylgalactosamine. The composition of the lipopolysaccharides was more complex than that of the exopolysaccharides and, in addition to the neutral hexoses and amino sugars, rhamnose was identified in two preparations and ribose in another. No lipopolysaccharide preparations contained O-methyl xylose or heptose.

The polysaccharides secreted by the bacillary forms grown on solid or in liquid media closely resembled the polysaccharides isolated from the fruiting bodies, in which they provided a matrix surrounding the microcysts. Each pair of polysaccharides contained the same monosaccharides, although in slightly different proportions. Differences were found in preparations from different strains. These results suggest that in the developmental cycle of the genus Myxococcus, considerable use is made of pre-existing enzyme systems to synthesize the precursors necessary for polysaccharide synthesis. Any specific difference between the polysaccharide produced by the bacilli and that surrounding the microcysts may lie in the fine structure, rather than in the individual components.

SUMMARY: Preparations of chitin synthetase (EC. 2.4.1.16) from the stipe of the toadstool Coprinus cinereus were characterized. Microsomal fractions had a very high specific activity. The chitin synthetase had been solubilized by treatment with digitonin, with an increase in specific activity and in stability. Optimum conditions for enzyme activity were determined. These preparations needed only a divalent cation and UDP-N-acetylglucosamine for chitin synthesis and showed no primer requirement. The sole glycosyl product was characterized as macro-molecular chitin.

SUMMARY: The solubilized chitin synthetase preparation from the stipes of Coprinus cinereus is activated by its substrate, UDP-N-acetylglucosamine, and by carbohydrates, in particular N-acetylglucosamine, diacetylchitobiose and glucose. Analysis of the results of variation in enzyme activity with UDP-GlcNAc concentration by several methods suggested that this substrate is an allosteric activator, with Hill coefficients close to 2 and 4 at concentrations above and below 0·1 mm respectively, and an [S]0·5 value of 0·9 mm. N-acetylglucosamine lessened but did not abolish the sigmoidal shape of the velocity-substrate concentration plot. The chitin synthetase was inhibited by adenine and uridine nucleotides, and uridine diphosphate was a powerful competitive inhibitor. The enzyme preparation also contained a nucleoside diphosphatase activity and the implications of the removal of the inhibitory UDP formed by the chitin synthetase are considered.

SUMMARY: Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80 % of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KC1 and Mg2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.

SUMMARY: A bacterium which can rapidly degrade phospholipids has been isolated from the ovine rumen and tentatively identified as a non-cellulolytic strain of Butyrivibrio fibrisolvens. When the organism was grown in batch culture the phospholipase activity appeared during the late exponential phase, peaked 4 to 8 h after maximum culture turbidity had been reached, and then rapidly disappeared. Activity was associated both with the whole cells and the cell-free culture supernatant.

Phosphatidylcholine was degraded via lysolecithin to produce free fatty acids and glycerylphosphorylcholine, indicating phospholipase A + B activity. The activity was stimulated by the addition to the incubation medium of oleic acid or sodium dodecylsulphate. There was no requirement for divalent cations, but the presence of cysteine at 10−2 m stimulated the activity many-fold even though the incubation was performed under an oxygen-free gas phase. Phosphatidylethanolamine and phosphatidylinositol were also attacked. The possible role of the organism in bringing about the degradation in the rumen of plant phospholipids in the sheep's diet is discussed.

SUMMARY: The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.

SUMMARY: The location of antigens on sections of bacteria using the soluble peroxidase-anti-peroxidase complex in conjunction with unlabelled antibody is described. Using this technique, spore antigens have been detected in the cytoplasm of vegetative cells during forespore septum formation and subsequent stages of sporulation. Antigenic sites were first associated with poly-beta-hydroxybutyric acid granules and subsequently were found in increasing quantities in the cytoplasm of the sporangium. Vegetative cell antigens were located on the cell wall and in the cortical region during sporulation. During germination antigens were located in the cortical region, and during outgrowth on the cell wall. These findings are discussed in the light of existing biochemical data.

SUMMARY: One induced and one spontaneous serum-resistant mutant were derived from smooth serum-sensitive Escherichia coli strains. There was little difference in the yield or the O-side-chain-sugar to core-sugar ratio of lipopolysaccharides from the mutants compared with the parental strains. The mutations to serum resistance were not accompanied by increases in either the amount or haemagglutination-inhibiting activity of acidic polysaccharides extracted from the strains.

Two colonially rough forms, designated 17fa and 17gb, were isolated from an aged culture of serum-resistant mutant 17. Escherichia coli 17fa appeared to be a som mutant and was rapidly killed by serum. Escherichia coli 17gb retained sero-logical O-specificity, and 17gb lipopolysaccharide contained a full complement of O-side-chains; the strain was killed by serum but only after a delay of 1 h.

Escherichia coli K-negative O8 donor Hfr59, which was serum-resistant, was crossed with rough serum-sensitive E. coli strain F470 and his + recombinants were selected. The majority of his + recombinants inherited a full complement of lipo-polysaccharide O-side-chains but were killed by serum after a delay of 1 to 2 h.

These results suggest that lipopolysaccharide O-side-chains are responsible for a delay in the killing by normal human serum of smooth E. coli strains but that other factors determine full serum resistance. No evidence was found of a role for K-antigens in serum resistance.

SUMMARY: Because the cot-2 and inv loci of Neurospora crassa are closely linked, the invertase from the morphological mutant, cot-2, was examined. The cot-2 strains produce an invertase with altered heat sensitivity, K m, and ratio of heavy to light forms. The cellular localization of cot-2 invertase is different from that of the wild type. There were no observable changes in the energy of activation or the pH optimum of cot-2 invertase, and some of the differences detected were not apparent under culture conditions that promoted wild-type growth. Since recombination (about 5 %) occurred between cot-2 and inv and culture conditions affected the enzyme characteristics, we suggest cot-2 determines, in part, the carbohydrate composition of the enzyme.

SUMMARY: Studies of the Sendai virus haemagglutinin receptor on the human erythrocyte surface have confirmed that it involves 2→3 linked sialic acid. Because the primary specificity of Vibrio cholerae neuraminidase is for this linkage, it is able to compete with the virus for the receptor, to which it adsorbs strongly at low temperatures. Corynebacterium diphtheriae neuraminidase, whose principal specificity is for a sialic acid linkage other than 2→3, does not easily remove Sendai virus receptors, nor does it adsorb to the erythrocyte surface. A new definition of the term ‘receptor-destroying enzyme’ is given which takes both enzyme and virus specificity into account, and a modified assay method is suggested in order to overcome the problems due to enzyme adsorption.

SUMMARY: Sera of rabbits immunized with pili or formalized cultures of Neisseria gonorrhoeae contained pili antibodies. The reaction between pili and specific gamma globulin was followed by direct visual observation with an electron microscope. Using pili from 31 strains and antisera against three strains, only a few cross-reactions between pili were observed. From this it is concluded that gonococcal pili are antigenically heterogeneous. However, serum raised with a T3 culture (with no detectable pili) contained antibodies against pili of the homologous T2 strain. It is proposed that the pilus antigen may exist in a form different from the typical pilus in gonococci.

SUMMARY: Groups of chickens were vaccinated intravenously with live cultures of Escherichia coli k12 possessing transfer factors F, I, A2 or no transfer factor (Tra−) and their immunity challenged by injecting them intravenously with an F+ strain of Salmonella heidelberg or an F+ or I+ strain of Salmonella typhimurium. Compared with the Tra− strain, vaccination with the F+, but not the I+ or A2+, k12 strain significantly reduced the mortality rate caused by the F+ salmonella strains; vaccination with the I+ or A2+, but not the F+, k12 strain significantly reduced the mortality rate caused by the I+ salmonella strain.

SUMMARY: The effects of purified oxyleghaemoglobin added to suspensions of bacteroids prepared anaerobically from soybean root nodules, were studied in terms of uptake of dissolved O2, nitrogenase activity and dissolved O2 concentration, in the absence of a gas phase. Leghaemoglobin allowed maximum rates of O2 uptake to continue to a much lower range of concentrations of free dissolved O2 than in the absence of the protein. This effect was diminished when the leghaemoglobin concentration was less than about 50 μ m. Nitrogenase activity at a given O2 concentration was not increased by raising the leghaemoglobin concentration above about 50 μ m. The fractional oxygenation of leghaemoglobin giving half the maximal O2 consumption rate by bacteroids was usually about 0·2; with limiting leghaemoglobin concentrations it was higher. Rates of nitrogenase activity were invariably greater during periods when the discharge of O2 from oxyleghaemoglobin was occurring at the maximum rate, than when similar maximum O2 uptake rates were being supported by higher concentrations of free dissolved O2. When myoglobin was used as the O2 carrier protein in similar experiments, enhanced nitrogenase activity also accompanied the maximum rate of discharge from the carrier. This occurred at about four times the concentration of free dissolved O2 at which it occurred when leghaemoglobin was the carrier. These results are discussed in relation to current theories about the mechanism of leghaemoglobin action.