- Volume 134, Issue 5, 1988
Volume 134, Issue 5, 1988
- Biochemistry
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Characterization of Glycoside and Polysaccharide Hydrolases Secreted by the Rumen Anaerobic Fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis
More LessThe rumen anaerobic fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis were grown in the presence of cellulosic substrate (sisal fibres) and the properties of the glycosidases and polysaccharide-degrading enzymes produced by the organisms were studied. β d-Glucosidase (EC 3.2.1.21), β d-fucosidase (EC 3.2.1.38), β d-galactosidase (EC 3.2.1.23), β1,3-glucanase (EC 3.2.1.6), β1,4-glucanase (EC 3.2.1.4) and βxylanase (EC 3.2.1.8) had pH optima of 6.0 and temperature optima under the conditions of assay of 50–55 °C, whereas for βxylosidase (EC 3.2.1.37) the optima were 6·5 and 39 °C respectively. The apparent K m values of individual enzymes secreted by the three fungi were similar. The results show that S. communis, P. communis and N. frontalis produce the same range of enzymes, with similar properties, able to degrade cellulose and hemicellulose.
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Secretion Mechanism of Bacillus subtilis Levansucrase: Characterization of the Second Step
More LessThe kinetics of levansucrase secretion were examined in a strain of Bacillus subtilis which overproduces the enzyme (sacU h). Pulse-labelling experiments indicated that the second step of the levansucrase secretion process has the properties of a membrane active-transport system. This event appears to be directly linked to the influx of iron into the bacteria. The response of B. subtilis to the inducer of levansucrase synthesis was modulated by ferrichelators and the extent of the response varied with the nature of the ferrichelator. Ferridihydroxybenzoate markedly shortened the induction lag period. It is inferred from these data that iron occurs as a cofactor for components of the membrane sites of synthesis/secretion of B. subtilis levansucrase.
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Properties of a Conidial-bound Cellulase Enzyme System from Trichoderma reesei
More LessConidia of Trichoderma reesei QM 9414 are able to degrade crystalline cellulose to glucose in the absence of protein synthesis, indicating the presence of a cellulase enzyme system. Measurement of enzyme activities revealed the presence of several glycanases and glycosidases. The cellulase enzyme system appeared irrespective of the conditions applied to induce conidiation. Removal of the endo-1,4-β-glucanase from the cellulase system could be achieved by treatment of conidia with β-octylglucoside or Triton X-100. Conidia treated in this way grew poorly on cellulose but well on glycerol. The endo-1,4-β-glucanase released from conidia by β-octylglucoside appeared as a single enzyme upon SDS-PAGE/immunoblotting with an apparent molecular mass of 68 kDa, an isoelectric point of 4·8–5·3, and a pH optimum between pH 4–6. These properties were similar to endo-1,4-β-glucanase I purified from culture filtrates of cellulose-grown T. reesei. Thermostability of the released enzyme was, however, lower than that of endo-1,4-β-glucanase I. Germination of T. reesei conidia in a medium containing glycerol as a carbon source led to a loss of endo-1,4-β-glucanase activity which paralleled the decrease in the number of ungerminated conidia.
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- Development And Structure
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Localization by Electron Microscopy of Alkylsulphatases in Bacterial Cells
More LessAlkylsulphatases have been localized in cells of two bacterial isolates using transmission electron microscopy. Cells were incubated with the appropriate alkyl sulphate ester in the presence of Ba2+ ions. Inorganic sulphate liberated by alkylsulphatases was precipitated at the site of liberation as BaSO4. Electron microscopy of thin sections was used to locate electron-dense grains which were identified by energy dispersive analysis of X-rays (EDAX) as BaSO4. The long-chain primary alkylsulphatases of the detergent-degrading bacterium Pseudomonas C12B, active on C6-C14 primary alkyl sulphates, were located on the outer cell-wall. There was no activity inside the cells. In contrast, the short-chain (C3-C7) alkylsulphatase in a coryneform isolated for its ability to grow on but-1-yl sulphate was located entirely in the cytoplasm. The butylsulphatase was apparently associated with granules of poly-β-hydroxybutyric acid. The different locations for the long- and short-chain alkylsulphatases may be related to the relative potential toxicities of their ester substrates.
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Colicin K Decreases the Density of Intramembrane Particles (IMP) in the Cell Membrane of Escherichia coli
More LessToxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58–161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1–13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.
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Activation of β-Tubulin Gene during Early Development of the Plasmodium in Physarum polycephalum
More LessUninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodial transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific β2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50–60% committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0·1% of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodial transition immediately before or during the commitment event. Small amounts of β2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus β2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.
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- Ecology
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Survival of Halobacteria Within Fluid Inclusions in Salt Crystals
More LessWhen sodium chloride crystallizes in an evaporitic environment, living halobacteria are entrapped within the fluid inclusions which form as the crystals develop. Trapped cells have been observed in natural salts from a marine saltern and from Lake Magadi. Entrapment occurs under both neutral and alkaline conditions. Salt crystals have been grown under controlled conditions from solutions containing pure culture suspensions of halobacteria at densities comparable to those reported from natural evaporitic habitats. Crystals formed in solutions heavily loaded with bacterial cells contained more and larger inclusions than crystals formed from sterile solutions, and thus bacterial entrapment may affect the physical characteristics of the product. Representative strains of each major grouping within the Halobacteriaceae except the genus Halococcus exhibited entrapment and survival within salt crystals. Continued motility has been demonstrated for up to three weeks after entrapment. All strains tested to date retained viability for a minimum of six months. Non-motile and non-viable cells were also entrapped.
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- Genetics And Molecular Biology
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The Nucleotide Sequence of the LPD1 Gene Encoding Lipoamide Dehydrogenase in Saccharomyces cerevisiae: Comparison between Eukaryotic and Prokaryotic Sequences for Related Enzymes and Identification of Potential Upstream Control Sites
More LessThe complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established. The flanking region 5′ to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes. The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase. The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.
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Cloning and Expression in Escherichia coli of a rec A-like Gene from the Acidophilic Autotroph Thiobacillus ferrooxidans
More LessA recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent M r equal to the higher-M r band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.
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Genetic Analysis of the Mycobacillin Biosynthetic Pathway in Bacillus subtilis B3
More LessTwelve mycobacillin-negative (My−) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. In whole-cell fermentations of some of these My− mutants a penta- and a nonapeptide accumulated; these peptides were also obtained in a cell-free system in which a new tripeptide was also detected. The amino acid composition, N- and C-terminal residues and amino acid sequence of these peptides agreed with those of equivalent segments of the mycobacillin molecule. The mycobacillin-synthesizing enzyme can be divided into three fractions that catalyse different steps in biosynthesis, and the defective enzyme fractions in the various mutant strains were identified by reconstitution experiments in vitro. The defects were further pin-pointed in mutant enzyme fractions by an ATP⇌Pi exchange reaction and also by cell-free synthesis involving the use of membrane-bound enzyme. The defects so identified indicated the formation of tri-, penta- and nonapeptides as intermediates in the mycobacillin biosynthetic pathway.
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Membrane Binding and Release of Bacillus subtilis DNA as a Function of the Cell Cycle
More LessThe presence of origin-region DNA in preparations containing bacterial cell wall and cytoplasmic membrane is well established, but little is known about the relationship between this association and events of the cell cycle. We have observed, during renewed growth of stationary-phase cultures of Bacillus subtilis, an association of DNA, including newly synthesized regions, with a specific region of the plasma membrane. Attachment was transitory, occurring once per replication cycle, and was prevented by inhibitors of cell wall synthesis.
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The Relationship between the Base Composition of Bacterial DNA and Its Intracellular Melting Temperature As Determined by Differential Scanning Calorimetry
More LessThe correlation between the melting temperature of intracellular DNA, determined by differential scanning calorimetry (DSC) of whole bacteria, and its guanine + cytosine (G + C) content, was examined for 58 species of bacteria. Samples of vegetative cells were heated in a Perkin-Elmer DSC-2C at 10 °C min-1 from 5 to 130 °C, cooled to 5 °C and then re-heated as before. Literature values for the mole fraction of G + C, X GC, were linearly related to the temperature, T max, at which the reversible peak, p r, observed on the second heating run was at a maximum, via the equation X GC = (T max − 73·8)/41·0. This equation accounted for 91·9% of the variance in X GC with 95% confidence limits of ±7·3%, approximately 1·6 times the corresponding uncertainty (±4·5%) quoted by De Ley (Journal of Bacteriology 101, 738–754, 1970) for estimates based on the spectroscopically determined melting temperature of purified DNA. Random errors of measurement of T max did not greatly limit the precision of the prediction and it was concluded that factors additional to base composition affected the temperature of DNA melting within the bacterial cell. Displacement of T max values from the fitted line was particularly noticeable in Campylobacter, Corynebacterium and Bacterionema species and part of the residual variation appeared to be species specific, possibly caused by differences in intracellular solute concentration.
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Transformation of Streptococcus sanguis to Intrinsic Penicillin Resistance
More LessA series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.
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Reversion from Erythromycin Dependence in Escherichia coli: Strains Altered in Ribosomal Sub-unit Association and Ribosome Assembly
More LessA mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. Strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.
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Characterization of B278, a Phage Different from Mu That Also Produces Auxotrophic Mutations in Escherichia coli K12
More LessBacteriophage B278 has been characterized and compared with Mu, the only phage known to produce random mutations in E. coli. Although both phages are morphologically indistinguishable and have a similar host range, they clearly differ at both the protein and the DNA level. B278 apparently possesses a DNA protection mechanism that is different from the mom system described for Mu.
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New Bacteriophages Active on Strains of Hyphomicrobium
Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (T m) ranged from 67 to 93 C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.
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Biochemical Analysis of Spontaneous fepA Mutants of Escherichia coli
More LessThe fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin. Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions. In this work seven spontaneous fepA mutations were analysed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies. In two strains, UT500 and UT700, the mutations were confined to the fepA gene. In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost. The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome. A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected. The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation. Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.
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- Immunology
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Immunological Properties of the Primer-independent Glucosyltransferase of Streptococcus mutans Serotypes d and g
More LessStreptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.
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- Pathogenicity And Medical Microbiology
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Cell Wall Components of Candida albicans as Immunomodulators: Induction of Natural Killer and Macrophage-mediated Peritoneal Cell Cytotoxicity in Mice by Mannoprotein and Glucan Fractions
Cell wall components from Candida albicans were compared to intact cells for their ability to induce natural cytotoxic immunoeffectors in the peritoneal cavity of mice. A soluble mannoprotein extract (MP) and an insoluble glucan fraction (GG) strongly stimulated the generation of peritoneal effectors capable of lysing YAC-1 and P-815 tumour cell lines in vitro. The anti-YAC-1 effectors were characterized as natural killer (NK) lymphocytes while the anti-P-815 effectors appeared to be activated macrophages. The activity of each fraction was typically dose-dependent and both fractions differed from whole cells in the kinetics of induction of cytotoxicity. However, the NK and macrophage effectors generated by these materials had similar functional and phenotypic properties, irrespective of the material used as inducer. No mannoprotein was detected in the insoluble glucan fraction GG. Hence, the immunoenhancing activity of GG could not be attributed to the presence of some MP or MP-like component. Mannan-rich fractions with low (<3%) protein content (M) or extracted by hot alkaline reagent (M-alk) were inactive as NK and macrophage inducers. Thus, the cell wall of C. albicans contains at least two distinct macromolecular complexes which mediate the induction in murine peritoneal exudates of cytotoxic effectors active against tumour cell lines.
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Demonstration of Two Protein Kinases in Extracts of Legionella micdadei
More LessProtein kinases I (PK I) and II (PK II) were purified 253- and 13·5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a K m of 2·7 mg ml−1 for PK I and 2·9 mg ml−1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6·8 and 7·0 for PK I and PK II respectively. The K m for ATP was 0·29 mm for PK I and 0·33 mm for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.
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Antibodies to the C Epitope of Neisseria gonorrhoeae Are Present in Patients with Gonorrhoea and Absent in Normal Sera
More LessWe have previously described a surface oligosaccharide antigen (epitope C) present in fresh isolates of Neisseria gonorrhoeae and in variants grown in subcutaneous chambers, but poorly formed by variants repeatedly subcultured in vitro. We have now investigated the presence of antibodies to epitope C in sera from normal individuals and from patients with gonorrhoea. Sera were analysed by Western blotting and ELISA, and compared with a pool of sera from normal individuals with no known history of gonorrhoea. Antigenic extracts and monoclonal antibody to the C epitope were used for competition and inhibition studies. Only the sera from patients contained antibodies to epitope C. Antibodies to several other gonococcal antigens were found in sera from patients, and also in normal sera. Collectively, the results indicate that epitope C is expressed in humans, that patients with gonorrhoea develop antibodies to it, and that such antibodies are absent in sera of normal individuals.
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Adherence of Streptococci to Surface-modified Glass
N. Satou, J. Satou, H. Shintani and K. OkudaFour types of surface-modified glass were prepared. Aminopropyl glass was prepared by alkylsilylation of glass slides with ?-aminopropyltriethoxysilane. This glass carries primary amino groups which may be protonated at pH 7.2. Owing to the presence of both positively charged ions and hydrophobic ethoxyl groups, the glass is considered to be amphipathic. Three other types of surface-modified glass slides were prepared from aminopropyl glass by forming Schiff's bases with three aldehydes: glucose, glyoxylic acid and hexanal. The aldehyde-treated slides were subsequently reduced using sodium borohydride. Thus, the surface of the glass was rendered hydrophilic, ampholytic or hydrophobic, respectively. The adherence of two Streptococcus sanguis strains and two Streptococcus mutans strains to the surface-modified glass slides was studied. Different strains showed differences in adherence to these slides depending on their physico-chemical surface properties. For S. sanguis ATCC 10556, hydrophobic bonds seemed to be most important, while in S. mutans OMZ 176, ionic interactions made the highest contribution to adhesion. Hydrogen bonds seemed to contribute least to adherence.
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Attachment of Mycobacteria to Fibronectin-coated Surfaces
This report investigates the extent of the expression of fibronectin (FN) binding properties among the mycobacteria and provides preliminary characteristics of the bacterial molecule(s) mediating attachment. Eight BCG substrains, three Mycobacterium tuberculosis strains and four other mycobacterial species all expressed FN-binding capacity. Treatment of organisms with detergent prior to the binding assay destroyed the FN-binding capacity of BCG but not that of Staphylococcus aureus. Trypsin pretreatment eliminated the FN-binding capacity of both BCG and S. aureus. [35S]Methionine-labelled material in supernatants from BCG and M. tuberculosis cultures attached to FN-coated surfaces. These culture supernatants inhibited the attachment of BCG but not S. aureus to FN-coated surfaces. This inhibitory activity of the supernatants was removed by affinity chromatography on FN-Sepharose but was not affected by similar passage over a control column (human serum albumin attached to Sepharose). These results demonstrate that the ability to bind FN is present in all mycobacterial species tested and suggest that attachment is mediated by trypsin-sensitive cell-surface component(s).
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HEp-2 Adhesion and the Expression of a 94 kDa Outer-membrane Protein by Strains of Escherichia coli Belonging to Enteropathogenic Serogroups
More LessSixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.
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- Physiology And Growth
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Cytoplasmic Alkalinization during Germ Tube Formation in Candida albicans
More LessWeak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6·8 to over 8·0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7·0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.
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Transcellular Ion Currents during Sporangium Development in the Water Mould Achlya bisexualis
More LessChanges in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the homogeneous stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.
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The Utilization of Pyridine Carbonitriles and Carboxamides by Nocardia rhodochrous LL100-21
More LessNocardia rhodochrous LL100-21 utilized 2-, 3- or 4-cyanopyridine (2-, 3- or 4-pyridine carbonitrile) and the corresponding pyridine carboxamides as sources of nitrogen for growth. Studies with intact bacteria and cell-free extracts indicated that 3-cyanopyridine was hydrolysed directly to nicotinic acid by an inducible 3-cyanopyridinase enzyme (a nitrilase), and that the organism also possessed a separately inducible nicotinamidase. In cultures supplied with 3-cyanopyridine or nicotinamide as the source of nitrogen the compounds were hydrolysed to form ammonia, which was utilized for growth, and nicotinic acid, which was not further metabolized and accumulated in the culture medium. In cultures containing 3-cyanopyridine, which had a bacteriostatic effect, little growth occurred until all the nitrile had been converted to nicotinic acid; the specific activity of 3-cyanopyridinase of the bacteria was highest in the early exponential phase of growth and had disappeared by the stationary phase. Measurements of the abilities to either oxidize or release ammonia from heterocyclic, aromatic and aliphatic nitriles and amides by bacteria grown on the various substrates indicated that benzonitrile acted as both a substrate and an inducer of 3-cyanopyridinase activity, whereas benzamide acted as a substrate and an inducer of nicotinamidase. These enzymes are induced separately from the acetonitrile hydratase/acetamidase enzyme system of this bacterium.
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Adaptation of the Kinetics of Glucose Transport to Environmental Conditions in the Yeast Candida utilis CBS 621: a Continuous-culture Study
More LessThe relation between the kinetic parameters of glucose transport and the physiology of Candida utilis CBS 621 was studied in chemostat cultures. In glucose-limited cultures the transport parameters were dependent on the growth rate of the yeast. Three different transport systems were found which differed by an order of magnitude in their affinity constants, namely a high-affinity (K m 25 µm), a medium-affinity (K m 190 µm), and a low-affinity uptake system (K m 2000 µm). Cells growing at a dilution rate of 0.45 h-1 or less had the high- and medium-affinity uptake systems. At a dilution rate of 0·52 h−1 the high-affinity system was absent and both the medium-and low-affinity systems were present. At a dilution rate close to µ max (0·57 h−1) only the low-affinity system was detected. The in situ contribution of each of the transport systems to glucose consumption in glucose-limited cultures was estimated on the basis of their kinetic parameters (K m and V max) and the residual glucose concentration in these cultures. The sum of the calculated rates of transport corresponded to the in situ rate of glucose consumption by the cultures as determined from the yield constant and the dilution rate. The dependence of the transport parameters on the growth rate and hence on the environmental sugar concentration was also evident in cells grown under nitrogen limitation. In contrast to carbon-limited cells, nitrogen-limited cultures growing at D = 0·15 h−1 did not exhibit the high-affinity glucose uptake system, whereas the medium- and low-affinity systems were present.
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Glutathione Formation in Penicillium chrysogenum: Stimulatory Effect of Ammonium
More LessPenicillium chrysogenum produced glutathione after growth in a defined medium containing 10mm-NH4Cl as the sole source of nitrogen. The use of higher ammonium concentrations (100 mm) resulted in stimulation of growth and glutathione formation. In addition, increases in the intracellular pools of glutamate, alanine and glutamine, proportional to the amount of ammonium present in the medium were observed. Resting cell systems, prepared from cells previously grown with ammonium, were able to produce glutathione when incubated with ammonium or the amino acids glutamate, alanine and glutamine. A mutant lacking NADP-dependent glutamate dehydrogenase activity (which has a leaky phenotype on ammonium as sole nitrogen source) required glutamate to synthesize glutathione. Resting cell systems of this mutant, prepared from cells previously grown with ammonium, did not produce glutathione even when incubated with glutamate or glutamine. On the other hand, resting cell systems of this mutant produced glutathione if prepared from cells previously grown with glutamate. The addition of glutamate to resting cell systems of the wild-type strain stimulated the synthesis of γ-glutamylcysteine synthetase, the first enzyme of glutathione biosynthesis.
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Ethanol Tolerance and the Induction of Stress Proteins by Ethanol in Candida albicans
More LessEthanol is one of the products of the metabolism of glucose by Candida albicans. The amount produced is directly related to the concentration of glucose in the medium. The fungus utilizes ethanol as a sole source of carbon but is relatively intolerant of ethanol in its environment. Ethanol induces germ tube formation by blastoconidia of C. albicans. Germination was not seen under fermentation conditions even though the amount of ethanol produced was in the range that induced germ tubes when added to phosphate buffer. Ethanol also induces C. albicans to form stress proteins that are similar to heat shock proteins. The possibility that stress proteins may regulate germ tube formation by C. albicans is discussed.
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Purification, Some Properties and Possible Physiological Role of an Extracellular Cobalamin Binding Protein from Euglena gracilis
More LessThe extracellular cobalamin (Cbl) binding protein from Euglena gracilis was purified and some properties of the protein were studied for the elucidation of its physiological role. The protein was purified about 20-fold with a yield of 15% and was homogeneous on PAGE. SDS-PAGE indicated that the protein had a single type of polypeptide of M r 56000. The protein could bind some Cbl analogues with different β-coordination moieties, over a wide range of pH values from 4·0 to 9·0, and the K s value for cyanocobalamin was 1·1 nm. The extracellular Cbl binding protein was located on the cell surface of E. gracilis, probably bound in the muciferous layer.
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- Systematics
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Taxonomic Studies on Brochothrix, Erysipelothrix, Listeria and Atypical Lactobacilli
More LessOne hundred and eighty-seven strains of listeriae, atypical lactobacilli, Brochothrix, Erysipelothrix and related Gram-positive bacteria were tested for 140 characters based on morphology, physiology and biochemistry. Computer analyses of the data resulted in the recovery of 20 phenons. Representative strains were examined for cytochrome content, cell wall, fatty acid and isoprenoid quinone composition and the G + C content of the DNA. The results indicate that the species Listeria monocytogenes, L. innocua and L. ivanovii are phenotypically very similar. L. grayi and L. murrayi are more distinct from these but are so similar to each other that L. murrayi should be regarded as a subspecies of L. grayi. Some of the atypical lactobacilli studied are members of the genus Brochothrix; the others probably represent four distinct species in a new genus.
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DNA Probes with Different Specificities from a Cloned 23S rRNA Gene of Micrococcus luteus
More LessA 7500 bp PstI restriction fragment of chromosomal DNA from Micrococcus luteus containing a 23S rRNA gene was cloned in vector pHE3 in E. coli RR 28 (the recombinant plasmid was designated pAR1). A recombinant phage (pAR5) hybridizing to all eubacteria tested was constructed by shotgun subcloning of the PstI fragment in phage M13mp8. Further subcloning of the fragments of the 23S rRNA gene in the vectors pTZ18R and pTZ19R using selected restriction sites of the gene enabled us to select cloned fragments of the 23S rRNA gene representing different specificities. Probes specific for Micrococcus luteus-Micrococcus lylae (pAR28), for the Arthrobacter-Micrococcus group (pAR27), for eubacteria (pAR5), and for the detection of eu- and archaebacteria (the so-called universal probe pAR17) were constructed. The specificity of each probe was analysed by dot hybridization to the chromosomal DNAs of representatives of most of the main phyla of eu- and archaebacteria.
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- Corrigenda
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