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Volume 128,
Issue 7,
1982
Volume 128, Issue 7, 1982
- Biochemistry
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Evidence for the Presence of Two Terminal Oxidases in the Trypanosomatid Crithidia oncopelti
C. Edwards and B. ChanceIncreasing concentrations of cyanide inhibited the respiration of whole cells of Crithidia oncopelti in a biphasic fashion. Approximately 80% inhibition was attained with 40 μm-KCN. No further inhibition occurred until the concentration of KCN reached approximately 200 μm. Thereafter inhibition rose gradually to 100% at 1500 μm-KCN. Difference spectra revealed the presence of two CO-reacting haemoproteins. These were shown to be two different functional oxidases by photochemical action spectra obtained by using laser light. One oxidase was identified as cytochrome a+a 3 whilst the other had the properties of cytochrome o. Both oxidases could be detected in cells at all stages of growth by the above methods.
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Characterization of the Assimilatory and Dissimilatory Pathways of Carbon Metabolism during Growth of Methylophilus methylotrophus on Methanol
More LessThe enzyme profile of methanol-grown Methylophilus methylotrophus has been determined. It shows that the organism uses a variant of the ribulose monophosphate cycle of formaldehyde fixation that involves cleavage of hexose phosphate by 2-keto-3-deoxy-6-phosphogluconate aldolase and a rearrangement sequence involving transketolase and transaldolase. The organism possesses high concentrations of a glucose-6-phosphate dehydrogenase active with both NADP+ and NAD+, and two separate 6-phosphogluconate dehydrogenases, one active with both NADP and NAD+ and the other active only with NAD+. In addition, the organism contains methanol dehydrogenase, and NAD+-linked formaldehyde and formate dehydrogenases, thus possessing the enzymic potential necessary for both cyclic and linear sequences for oxidation of the formaldehyde derived from methanol. Hexulose phosphate synthase, phosphohexuloisomerase, glucose-6-phosphate dehydrogenase and the two 6-phosphogluconate dehydrogenases have been purified, characterized and examined for possible regulatory properties.
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A Possible Alternative Mechanism for the Oxidation of Formaldehyde to Formate
More LessA survey of the occurrence of a number of enzymes possibly involved in the C1 dissimilatory pathway in organisms using the serine pathway of carbon assimilation indicated that neither the methanol dehydrogenase nor the NH4 +-independent dye-linked formaldehyde dehydrogenase had a specific role in the oxidation of formaldehyde. Conversely, the activities of enzymes involved in the methenyl-THF pathway were shown to be induced during growth on methanol or methylamine, suggesting a potential dissimilatory role for this pathway.
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The Degradation of p-Coumaryl Alcohol by Aspergillus flavus
More LessAspergillus flavus utilized p-coumaryl alcohol, one of the major constituents of lignin, as sole carbon source. The following compounds were isolated from the growth medium and identified by means of their melting points, IR, NMR and mass spectra: p-coumaric acid, p-hydroxybenzoic acid and protocatechuic acid. Culture filtrates from mycelium grown on p-[1-14C]coumaryl alcohol contained p-[14C]coumaric acid, β-hydroxy-(p-hydroxyphenyl)[14C]propionic acid, (p-hydroxybenzoyl)[14C]acetic acid and [14C]acetic acid. Oxidation of protocatechuic acid by crude cell-free extracts formed β-ketoadipic acid, which was isolated and characterized.
A pathway for the degradation of p-coumaryl alcohol by Aspergillus flavus is proposed.
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Cross-pathway Control of Ornithine Carbamoyltransferase Synthesis in Neurospora crassa
More LessThe pattern of cross-pathway regulation of the arginine synthetic enzyme ornithine carbamoyltransferase was investigated in Neurospora crassa, using single and double mutant auxotrophic strains starved for their required amino acids. These experiments show that starvation for histidine, tryptophan, isoleucine, valine or arginine can result in derepression of ornithine carbamoyltransferase. Methionine starvation also gave slight derepression, but starvation for lysine or leucine gave little or no effect.
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Coordinate Production of Three Exoenzymes by Staphylococcus staphylolyticus
More LessStaphylococcus staphylolyticus produced three exoenzymes (a staphylolytic endopeptidase, a hexosaminidase and a protease) coordinately under a range of conditions of induction and repression by various peptides and carbohydrates. Mutants of S. staphylolyticus were isolated and shown to have pleiotropic variations in the production of the three enzymes. Hypo-or hyperproducing mutants of one enzyme were invariably hypo-or hyperproducers for the other two enzymes. Mutants that had lost the ability to produce one of the exoenzymes invariably failed to produce the other two enzymes. Revertants isolated from non-producers that regained the ability to produce one of the exoenzymes always regained the ability to produce the other two as well. These results suggest that the three exoenzymes share a common regulatory or processing mechanism.
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Purification of Pili and Outer Membrane Vesicles of Neisseria gonorrhoeae by Wheat Germ Agglutinin Affinity Chromatography
More LessPrevious studies indicated that OMVs of Neisseria gonorrhoeae reacted specifically with WGA. A technique was therefore developed for the separation of gonococcal pili and OMVs by WGA affinity chromatography. This method was more convenient and more effective than isopycnic centrifugation for obtaining OMVs free of pili. The antigens were further purified but though homogeneous as judged by in vitro analytical methods, they both elicited an antibody response in animals to minor impurities previously undetected.
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- Development And Structure
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Ultrastructure of the Cell Envelope Layers and Surface Details of Legionella pneumophila
More LessTen strains representing six serogroups of Legionella pneumophila were examined by electron microscopy using freeze-etching, thin-sectioning and negative-staining techniques. In addition, selected strains were examined further as shadowed and freeze-dried preparations and by scanning electron microscopy. The cell envelope consisted of two membranes, evident in fractured specimens as four short ridges. The major fracture plane was through the inner membrane, and therefore the protoplasmic and extracellular fracture faces of this membrane were predominant. With the exception of one strain (Togus 1), the particle arrangement on these fracture faces was random. A peptidoglycan-like cell wall layer was revealed only in sections of partially plasmolysed cells. Membrane-bounded poly-β-hydroxybutyrate-like granules were evident in the cytoplasm and these frequently showed plastic deformation due to fracturing. Although appendages were present, the surfaces of organisms and of isolated cell membranes showed no regular arrays of particles.
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Iron Transport in Mycobacterium smegmatis: The Location of Mycobactin by Electron Microscopy
More LessMycobactin, the lipid-soluble iron-binding compound of the mycobacteria, has been located using electron microscopy of whole cells stained with vanadate. It forms a discrete, discontinuous region close to, or even included in, the cytoplasmic membrane of Mycobacterium smegmatis. It does not occur throughout the whole thickness of the wall but is some distance within the envelope. Possible models for the accommodation of mycobactin are discussed. It is concluded from these observations that mycobactin may be acting either as a store of iron, or as an ionophore, or possibly fulfilling both roles.
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- Ecology
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Microbial Metabolism of Acetate, Propionate and Butyrate in Anoxic Sediment from the Colne Point Saltmarsh, Essex, U.K
More LessA gas chromatograph-gas proportional counting procedure has been developed to measure the in situ rates of metabolism of short-chain fatty acids in anaerobic saltmarsh sediment. The technique was used to determine the turnover rates of acetate, propionate and butyrate in sediment cores injected with 14C-labelled tracers and incubated at field temperature. Experiments showed that all three fatty acids were metabolized by micro-organisms present in the sediment, although an initial fast rate of disappearance of 14C from the injected [14C]propionate and [14C]butyrate was partly due to exchange of the tracer between an extractable pool of fatty acids and a tightly bound fatty acid pool which was not extracted. Measurement of the rates of metabolism of acetate, propionate and butyrate in intact cores of sediment from the surface layer (0–4 cm) of a saltmarsh pan showed that carbon flow through the acetate pool was far greater than that through either propionate or butyrate. Only about 14% of the acetate could have been derived from propionate and butyrate, 86% being from other precursors. The rate of acetate turnover decreased markedly with increased depth, and at 20 cm was negligible. The turnover of propionate and butyrate was totally inhibited by the presence of 20 mm-sodium molybdate, and their metabolism was attributed to the activity of sulphate-reducing bacteria.
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- Genetics And Molecular Biology
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Spontaneous Deletions in the TOL Plasmid pWW20 which Give Rise to the B3 Regulatory Mutants of Pseudomonas putida MT20
More LessThe size of the TOL plasmid pWW20 from Pseudomonas putida MT20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270–280 kilobase pairs (kb). During growth on benzoate, MT20 segregates strains carrying mutations in the plasmid regulatory gene xylS; these so-called B3 strains retain the ability to grow on m-xylene (Mxy+) but do not grow on its metabolite m-toluate (Mtol−) and have also lost the ability to transfer the plasmid (Tra−). Analysis of restriction digests of plasmid DNA from seven such segregants, independently isolated, showed that pWW20 had undergone extensive deletions of 90–100 kb. All the deleted plasmids had lost a common core of DNA, of about 72–80 kb, but in class A mutants the deletion extended at one end of this core and in class B mutants at the other end. Class A and B mutants also differed in their rate of growth on m-xylene as a result of differences in the level of expression of their plasmid-coded catabolic enzymes. This suggests that an additional gene, involved in regulating levels of gene expression, is located in the region uniquely deleted in the class B mutants.
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Thymine Metabolism in Pseudomonas aeruginosa Strain 1: The Presence of a Salvage Pathway
More LessExogenous thymine was found to be taken up very slowly by Pseudomonas aeruginosa in comparison to other pyrimidines, and most of it was catabolized by the cell. The existence of a functional, although inefficient, thymine salvage pathway was demonstrated and this pathway operated more effectively when de novo thymidine nucleotide biosynthesis was inhibited by trimethoprim or methotrexate. The mechanism of thymine salvage by P. aeruginosa appears to be different from that of Escherichia coli and Pseudomonas acidovorans as thymidine was not incorporated into the DNA. Like P. acidovorans, P. aeruginosa lacked thymidine phosphorylase activity. Unsuccessful attempts were made to isolate thymine auxotrophs.
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Cellulose-negative Mutants of Acetobacter xylinum
More LessCellulose-negative mutants of Acetobacter xylinum have been isolated. Chemical mutagenesis induced a high frequency of such mutants without significant cell killing. Cellulose synthesis could be activated phenotypically in the majority of the mutants by antibiotics that block RNA or protein synthesis. Therefore these mutations are not in the structural genes coding for the enzymes involved in cellulose synthesis, but their nature is not known. All of the spontaneous cellulose-negative mutants and the mutants induced by ethyl methanesulphonate or nitrous acid reverted when grown statically, but about 10% of the nitrosoguanidine-induced mutants did not.
Growth experiments with mixed cultures of the wild-type and cellulose-negative mutants were used to analyse the biological function of cellulose in A. xylinum. The cellulose-producing wild-type has a strong selective advantage compared with the cellulose-negative mutants when grown statically. This is in agreement with the hypothesis ( Schramm & Hestrin, 1954 ) that cellulose enables the cells to reach the surface of the liquid medium, where the supply of oxygen is abundant. Cellulose-negative mutants may be enriched in shake flask cultures because of selective aggregation of cellulose-producing cells.
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Effects of Anaerobiosis and Nitrate on the Expression of Succinate Dehydrogenase and Enzymes Associated with Nitrogen Metabolism in Klebsiella pneumoniae
More LessWe have shown that the low histidase activity found in anaerobic, nitrogen-limited cultures of Klebsiella pneumoniae is due to repression of the right-hand hut operon. In addition, we have examined the effects of NO3 − on the aerobic and anaerobic expression of catabolit- and NH4 −-repressible enzymes in this organism. NO3 − permitted anaerobic growth of K. pneumoniae in minimal medium containing histidine as the sole carbon source, and histidase and succinate dehydrogenase were derepressed during anaerobic growth in histidine/NO3 − medium. Use of sucrose rather than histidine as the carbon source reversed the effects of NO3 − and repressed histidase and succinate dehydrogenase activities. Anaerobic growth in sucrose/NO3 − medium also uncoupled the expression of urease and glutamine synthetase.
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Bacillus subtilis Extracellular Nuclease Production Associated with the spoOH Sporulation Locus
More LessA number of nuclease-deficient mutants of Bacillus subtilis were isolated and found to be concurrently asporogenous. The nuclease-deficient phenotype appeared to be associated with the spoOH sporulation locus. Spontaneously occurring sporogenous revertants concomitantly recovered the ability to produce extracellular nuclease activity. The position of the ncl mutation was determined using transformation and PBS1 transduction and found to map in the same site as spoOH. The map order of ncl and markers in the vicinity was determined to be purA-cysA-ncl(spoOH)-strA.
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- Immunology
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Structural and Functional Differences of the Anionic and Cationic Antigens in K99 Extracts of Escherichia coli B41
More LessRadiolabelled anionic and cationic components were purified from K99 extracts of Escherichia coli B41 by immunoelectrophoresis using absorbed K99 antisera. SDS-polyacrylamide gel electrophoresis revealed that the apparent molecular weights of the polypeptide subunits were 34 000 and 19 000, respectively. Both anionic and cationic antigens in cell-free K99 extracts adhered to sheep erythrocytes after 90 min at 4C, but the cationic antigen eluted after a further 1 h at 37 °C. The anionic antigen did not elute from sheep erythrocytes after 18 h at 37, 43 or 56 °C.
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- Pathogenicity And Medical Microbiology
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Accessibility of Enterobacterial Common Antigen to Antibodies in Encapsulated and Non-capsulated S and R Forms of Escherichia coli
G. Acker, G. Schmidt and H. MayerAntiserum specific for the enterobacterial ‘common antigen’ (ECA) was obtained by absorbing a rabbit ECA antiserum with an ECA-negative mutant. Accessibility of ECA to antibodies in encapsulated and non-capsulated S and R forms derived from Escherichia coli O8: K27 was studied using the indirect immunoferritin technique (whole-mount electron microscopy). The number of ferritin particles on the bacterial surface decreased in the order, non-capsulated R > encapsulated R > non-capsulated S > encapsulated S form, indicating that both the O and K antigens partly cover ECA on the surface of the outer membrane. Whole-mount and thin-section electron microscopy showed that ferritin was evenly distributed on the surface of R mutants, whereas it formed clusters on the S forms.
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- Physiology And Growth
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Production and Tolerance of Ethanol in Relation to Phospholipid Fatty-acyl Composition in Saccharomyces cerevisiae NCYC 431
More LessAccumulation of ethanol in supernatants from anaerobic cultures of Saccharomyces cerevisiae NCYC 431 closely paralleled growth during the early exponential phase of batch growth, and continued after growth had ceased. During the 8–64 h period of the fermentation, the intracellular ethanol concentration was greater than the extracellular concentration. Ethanol was very rapidly extracted from organisms by washing with water. During growth up to 32 h, there was a progressive decrease in fatty-acyl unsaturation in phospholipids, and a corresponding proportional increase in saturation. Thereafter, the trend was very slightly reversed. Supplementing cultures with ethanol (0·5 or 1·0 m) after 8h incubation retarded growth rate, while supplementation with 1·5 m-ethanol immediately stopped growth. In cultures supplemented with 0·5 or 1·0 m-ethanol, viability was not lowered, but supplementation with 1·5 m-ethanol caused a rapid decline in viability. Supplementation of cultures with ethanol at any of the three concentrations led to an increase in the proportion of mono-unsaturated fatty-acyl residues in cellular phospholipids, especially in C18 residues, which was accompanied by a decrease in the proportion of saturated residues.
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Selenium-dependent Growth and Glycine Fermentation by Clostridium purinolyticum
More LessClostridium purinolyticum fermented glycine as a sole carbon and energy source according to the equation:
4 Glycine + 2H2O → 3 Acetate + 2CO2 + 4NH3
The organism required adenine as a supplement and selenium compounds as micronutrients for growth. The molar growth yield on glycine was 6·5 g dry wt. Radiochemical and enzymic investigations revealed a new fermentation pathway for glycine in which 1 mol glycine was completely oxidized to CO2 and the generated reducing equivalents were used to reduce a further 3 mol glycine to acetate via the glycine reductase system. This reaction was associated with the formation of ATP.
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Sclerotization in Relation to Plasmodial Senescence in the Acellular Slime Mould Didymium iridis
More LessWhen subjected to dehydration, Didymium iridis plasmodia differentiate into sclerotia which can be maintained for long periods and exhibit no discernible O2 uptake. While actively growing plasmodia display the phenomenon of senescence, this ageing is delayed in sclerotia by the duration of the sclerotized state. Upon rehydration, plasmodia live the remainder of their characteristic life spans. Hence, the timing mechanism for senescence is conserved during this reversible differentiation pathway.
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