- Volume 135, Issue 2, 1989
Volume 135, Issue 2, 1989
- Biochemistry
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Site-directed Inhibition of Haemophilus influenzae Malate Dehydrogenase
More LessPrevious studies of Haemophilus organisms documented the importance of an NAD+-dependent malate dehydrogenase in the incomplete tricarboxylic acid cycle present in these organisms. Selective interactions occurring at the coenzyme and substrate binding sites of a purified Haemophilus influenzae malate dehydrogenase were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated the presence of regions in the coenzyme binding site that interacted with the adenosine and pyrophosphate moieties of the coenzyme. Positive chainlength effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region at this site that was close to the pyrophosphate region. Seven analogues of NAD+ that were structurally altered in either the pyridine or purine ring were evaluated as selective inhibitors of the enzyme. The three most effective inhibitors of the purified malate dehydrogenase inhibited the growth of H. influenzae when the organism was grown on a limiting concentration of NAD+.
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The Anaplerotic Phosphoenolpyruvate Carboxylase of the Tricarboxylic Acid Cycle Deficient Acholeplasma laidlawii B-PG9
More LessPhosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEP-C) was purified approximately 770-fold from the mollicute Acholeplasma laidlawii B-PG9. The partially purified PEP-C required phosphoenolpyruvate (PEP) and MnC12 at pH 7·4 or MgC12 at pH 8.6 for optimal activity. The product is oxaloacetate as detected by a malate dehydrogenase indicator system. The Km a (PEP variable) was 0.66 mm and the Km b (bicarbonate variable) was 1·02 mm. At low bicarbonate concentrations (0·5 mm), PEP-C activity was stimulated approximately 240% by fructose 1,6-bisphosphate. Aspartate was a non-competitive inhibitor of PEP-C activity. The Ki a (PEP variable) for aspartate was 0·69 mm and the Ki b (bicarbonate variable) was 0·99 mm. Malate, citrate, isocitrate, 2-oxoglutarate, acetyl-CoA, CMP, CDP, GDP, GTP, ADP and ATP had no effect on the PEP-C reaction. The Hill interaction coefficient was 0·98–1·11. The molecular mass by sucrose density gradient analysis was 353 kDa; by gel filtration chromatography it was 384 kDa. The Stokes radius was about 7·4 nm. PEP-C activity and its inhibition by aspartate in Acholeplasma laidlawii B-PG-9 extracts may reflect an involvement of this enzyme in the interdependent regulation of protein, lipid and nucleic acid precursor metabolism of this TCA-cycle-deficient and cytochrome-less mollicute.
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Characterization of Cell Wall Components of the Alkalophilic Bacillus Strain C-125: Identification of a Polymer Composed of Polyglutamate and Polyglucuronate
More LessThe A2 substance, a structural component of the cell wall of alkalophilic Bacillus strain C-125, has an M r of 21000 and is composed mainly of glucuronic acid and glutamic acid. Hydrazinolysis of the A2 substance yielded homoglucuronate with an estimated M r of 4800. The M r of polyglutamate previously prepared from the A2 substance by deglycosylation was estimated to be 14000. It is therefore suggested that the A2 substance is composed of the two polymers, polyglutamate and polyglucuronate, and the name ‘teichuronopeptide’ is proposed for this complex.
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Characterization of Purine Hydroxylase I from Aspergillus nidulans
More LessPurine hydroxylase I from Aspergillus nidulans was purified 850-fold. The purified preparations exhibited the spectral and catalytic properties, including broad specificity for oxidizing and reducing substrates, typical of molybdenum/flavin/iron-sulphur-containing hydroxylases (oxotransferases).
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Chitin Synthase Activity from Neocallimastix frontalis, an Anaerobic Rumen Fungus
More LessChitin synthase activity was detected in actively growing mycelium of Neocallimastix frontalis after mechanical disruption of the cells. Chitin formation in fungal extracts at 32 °C was linear with respect to time for at least 60 min, and with respect to protein concentration up to 750 μg ml−1. The optimum pH for enzyme activity was 8·5 using 10 mm-Tris/HCl buffer. Mg2+ was necessary for maximum activity and 10 mm-MgC12 was routinely used during the assays. The apparent K m for the substrate UDP-GlcNAc was 2 mm. Polyoxin D was a competitive inhibitor of chitin synthesis with an apparent K i of 4 μm. Following treatment with trypsin (12·5 μg ml−1), the chitin synthase activity of the fungal extract increased by six-fold, indicating that most of the chitin synthase activity was zymogenic. The reaction product was insoluble in 1 m-KOH or 1 m-acetic acid, but it was solubilized by heating in 6 m-HCl at 120 °C for 2.5 h and was hydrolysed by chitinase into diacetylchitobiose.
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Purification and Characterization of a Thermostable β-Xylosidase from Thermomonospora fusca
More LessXylan-degrading enzymes, including β-xylosidase (EC 3.2.1.37), were induced when Thermomonospora fusca was grown at 50 °C in liquid medium containing 0·2% xylan. The intracellular β-xylosidase activity was concentrated and characterized by fast protein liquid chromatography and gel electrophoresis. A zymogram technique was developed to identify β-xylosidase directly on polyacrylamide gels. A single enzyme (168 kDa; pI 4·37) was identified and purified to homogeneity. The consistent detection of a single band on denaturing SDS gels suggested that the enzyme was composed of identical subunits; since the subunit molecular mass was 56 kDa, a trimeric structure is suggested. High activity against p-nitrophenyl β-d-xylopyranoside (pNPX) occurred in the pH range 5·0–9·0 and temperature range 40–60 °C. The enzyme was stable at room temperature at pH 6·0–8·0; it had a half-life of 8 h at 65 °C, and of 1·5 h at 70 °C. The purified enzyme did not exhibit any detectable activity against arabinoxylan, carboxymethylcellulose or p-nitrophenyl β-d-glucopyranoside. The enzyme had a K m of 0·89 mm (pNPX) and was inhibited by d-xylose (K i 19 mm) but not d-glucose. The size of the T. fusca enzyme is in the range reported for the few other bacterial β-xylosidases described, but the acidic nature of the protein and its affinity for the substrate have more in common with some of the monomeric β-xylosidases described in fungi.
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O-Glycosylation of Proteins by Membrane Fractions of Trichoderma reesei QM 9414
More LessIn order to investigate O-glycosylation of proteins in the fungus Trichoderma reesei QM 9414, a membrane preparation was isolated and used to study the glycosylation of endogenous proteins. Exogenously added GDP-[U-14C]mannose was used to mannosylate both endogenous lipid and protein. The kinetics of mannosylation together with pulse-chase experiments with cold GDP-mannose revealed that lipid was labelled before protein. The lipid was identified as mannosyl phosphoryl dolichol (Dol-P-Man) by TLC together with an authentic standard from yeast. Addition of tsushimycin, a specific inhibitor of Dol-P-Man synthesis, completely blocked transfer of mannose from GDP-[U-14C]mannose to endogenous lipid. The mannosyl units transferred to endogenous protein could be released by β-elimination, and were shown to consist mainly of tetra-, di- and monomannosyl chains. Mannosylation of endogenous proteins occurred at a lower rate with membranes isolated from glycerol-grown cells. This could be overcome by addition of cold GDP-mannose, suggesting a limitation of endogenous GDP-mannose and/or dolichol phosphate in glycerol-grown (i.e. catabolite-repressed) cells.
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Purification and Some Properties of Candida albicans Exo-1,3-β-glucanase
More LessAn exo-1,3 β-glucanase was purified from blastoconidia of Candida albicans 1001. The purified enzyme appeared as a single protein band by PAGE, and split into two subunits (M r approximately 63000 and 44000) when analysed by SDS-PAGE. The pI of the enzyme was 4 and a K m of 1·7 mg ml−1 was estimated for laminarin as substrate. Despite its very reduced activity on the synthetic substrate p-nitrophenyl β-d-glucoside, C. albicans exo-1,3-β-glucanase hydrolysed 1,3-β-glucan by an exo-splitting mechanism and was inhibited by glucono-δ-lactone and by Hg2+ and Ag+ cations. The active exo-glucanase was mainly located in the periplasm, but it was also present inside the cytoplasmic membrane in small amounts and was secreted into the culture medium. The electrophoretic mobility of the enzyme from all three locations was the same.
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The Urease of Ureaplasma urealyticum
The urease from Ureaplasma urealyticum (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6·9 and pI values were determined. Storage of the purified enzyme at −70 °C followed by thawing to 20 °C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes on the enzyme, all distinct from its active site.
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Purification and Characterization of Extracellular Glucosyltransferase Synthesizing Water-insoluble Glucan from Streptococcus rattus
More LessAn extracellular glucosyltransferase synthesizing water-insoluble glucan (GTF-I) was purified from the culture supernatant of Streptococcus rattus strain BHT (mutans serotype b) by hydroxylapatite chromatography, DEAE-Toyopearl chromatography and preparative isoelectric focusing. The M r of GTF-I was 155000 by SDS-PAGE and the isoelectric point was pH 4·9. The specific activity, the optimum pH and the K m value for sucrose were 10·0 i.u. (mg protein)−1, 6·5 and 2·4 mm, respectively. The enzyme synthesized a water-insoluble glucan consisting of 69·4 mol% 1,3-α-linked glucose, 23·6 mol% 1,6-α-linked glucose, 2·4 mol% 1,3,6-α-branched glucose and 4·4 mol% non-reducing terminal glucose, and also a small amount (3% of the total glucan) of soluble glucan with 82·4 mol% 1,6-α-linked glucose. The M r and pI values of purified GTF-I were identical with those of the enzyme in the culture supernatant.
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Purification and Characterization of Cell-associated Glucosyltransferase Synthesizing Water-insoluble Glucan from Serotype c Streptococcus mutans
More LessStrains of Streptococcus mutans (serotypes c, e and f) were found to possess high levels of glucosyltransferase (GTase) activity, both cell-associated and in the culture medium, when grown in either sucrose-free or sucrose-containing broth media. The cell-associated GTase of S. mutans MT8148 (serotype c) was effectively extracted by treatment with 8 m-urea at 25 °C for 1 h. Approximately 95% of the GTase activity was solubilized by this treatment. The crude extract was purified by DEAE-Sephacel and hydroxylapatite column chromatography. For comparison, extracellular GTase was also purified from the culture supernatant of the same strain by ammonium sulphate precipitation, chromatofocusing and hydroxylapatite chromatography. The molecular masses of the cell-associated and extracellular GTase proteins were similar (156 kDa) as determined by SDS-PAGE. However, the pH optima for maximum GTase activity were different: pH 6·7 to 7·0 for the cell-associated enzyme and pH 5·5 to 6·5 for the extracellular enzyme. The product of cell-associated GTase from sucrose was almost exclusively water-insoluble glucan. On the other hand, extracellular GTase produced mainly water-soluble glucan from sucrose. This indicates that GTase synthesizing water-insoluble glucan is present primarily in a cell-associated form in serotype c S. mutans. Insoluble glucan synthesis by the cell-associated GTase from sucrose was not enhanced by addition of primer dextran T10 to the reaction mixture. The extracellular and cell-associated GTases were immunologically unrelated as determined by ELISA using monoclonal antibodies.
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Purification and Some Properties of a Novel l-2,4-Diaminobutyric Acid Decarboxylase from Vibrio alginolyticus
More LessPrevious investigations have shown that members of the genus Vibrio possess a novel enzyme activity decarboxylating l-2,4-diaminobutyric acid (DABA) to 1,3-diaminopropane (DAP). In this paper we describe the purification, by about 3600-fold, of the enzyme from V. alginolyticus. The purified enzyme was apparently homogeneous, and had a specific activity of 4200 nmol DAPmin−1 (mg protein)−1. The enzyme protein has an M r of 450000±20000 and is apparently comprised of four identical subunits (M r 109000±1000). Neither 2,3-diaminopropionic acid, ornithine, lysine nor arginine served as substrates. Some properties of the enzyme were determined. Cultivation of this bacterium in the presence of added DABA brought about increased production of norspermidine (NSPD), characteristically present in this species as well as DAP, suggesting that the enzyme may be functionally implicated in the formation of DAP, a biosynthetic precursor of NSPD.
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- Ecology
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Factors Affecting Conjugal Transfer of Plasmids Encoding Mercury Resistance from Pure Cultures and Mixed Natural Suspensions of Epilithic Bacteria
More LessSixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8·4 × 10−8 to 2·8 × 10−3 per recipient. Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range. Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10–25 ° C at 3·5 g C 1−1 and with donor to recipient ratios of 0·4–30. Transfer occurred over a range of pH values (pH 5·0–8·0) but the effect of pH was most significant at non-optimal temperature. Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3. Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp. on agar in the laboratory. Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 × 10−5 per recipient) after 24 h, at 25 °C, pH 5·5–8·0 and on a medium containing 10 g C1−1. The optimal initial cell density of MNS and recipient was 1·7 × 105 c.f.u. cm−2 and highest frequencies were obtained with donor to recipient ratios ranging from 1·2 × 10−1 to 1·7 × 10−3. Most of the plasmids (54%) from MNS transferred from their original P. aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1·1 × 10−6 to greater than 1·0 × 10−1 per recipient. The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance. Twenty-one plasmids > 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations. One of these alterations was associated with the acquisition of streptomycin resistance. Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions.
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- Genetics And Molecular Biology
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Characterization and Complementation of Mutants of Methylobacterium AM1 Which Are Defective in C-1 Assimilation
S. Stone and P.M. GoodwinWe investigated eight serine pathway mutants of Methylobacterium AM1; one lacked serine glyoxylate aminotransferase, four were defective in glycerate kinase, and three were blocked in the unknown part of the serine pathway which involves conversion of acetyl-CoA to glyoxylate. We complemented six of these mutants using a gene library of Methylobacterium AM1 DNA and identified two new regions of the chromosome which encode polypeptides necessary for C-1 assimilation. One is an 8·8 kbp HindIII fragment required for the production of glycerate kinase and the other a HindIII fragment of approximately 13 kbp essential for the conversion of acetyl-CoA to glyoxylate. These new C-1 regions of the chromosome are not closely linked to the previously described methanol oxidation genes, nor to a gene required for the production of an active malyl-CoA lyase. Our mutants were also complemented by clones from a gene library of a related pink pigmented facultative methylotroph, Methylobacterium organophilum.
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A New Regulatory Locus of the Maltose Regulon in Klebsiella pneumoniae Strain K21 Identified by the Study of Pullulanase Secretion Mutants
More LessThis study has shown that Klebsiella pneumoniae strain K21 differs from the previously characterized and closely related K. pneumoniae strain PAP996 in that expression of the pullulanase gene (pulA) and other genes of the maltose regulon is partially independent of exogenous inducer (maltose/maltotriose). Mutants of strain K21 which are defective in pullulanase synthesis and/or secretion were isolated following Tn10 mutagenesis. Three phenotypic classes of mutants were identified. Class I mutants were defective in the surface localization and secretion of pullulanase. Class II mutants did not secrete detectable levels of pullulanase but were able to export pullulanase to the cell surface. Class II mutants also expressed pullulanase and other maltose-regulated genes at markedly lower levels than those found in the parent strain under non-inducing conditions. The single class III mutant was intermediate between K21 and class I mutants; most of the cell-associated pullulanase was localized at the cell surface whilst a significant amount was secreted into the medium. Mapping indicated that all but three of the Tn10 insertions were adjacent to, and at either side of, pulA. One class II mutant carried a Tn10 insertion in or close to malT whereas in the remaining class II mutants the insertions were located at least 4 kb upstream of pulA in a region which may define a new regulatory locus of the maltose operon.
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High Frequency Variation of Colony Morphology and Chromosome Reorganization in the Pathogenic Yeast Candida albicans
More LessA clinical isolate of the pathogenic yeast Candida albicans varied in its colony morphology from smooth (o-smooth) to semi-rough type (SRT) and concomitantly lost its virulence for mice. In terms of DNA content, the smooth parent was near triploid when Saccharomyces cerevisiae strains of known ploidy were used as references. The SRT variant showed several features characteristic of polyploidy. From the SRT variant, revertant-like smooth (r-smooth) variants with recovered virulence were derived at a frequency of 5 × 10−3. The results of pulsed-field gel electrophoresis on chromosomal DNA showed changes in patterns of chromosome-sized DNA bands in the SRT variant as well as in r-smooth variants, which correlated with these variations. Correlations between colony morphology, state of ploidy and virulence of this asporogenous yeast are considered.
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- Pathogenicity And Medical Microbiology
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The Broad-specificity, Membrane-bound Lactate Dehydrogenase of Neisseria gonorrhoeae: Ties to Aromatic Metabolism
More LessNeisseria gonorrhoeae can convert phenyllactate (PL) to phenylalanine and 4-hydroxyphenyllactate (HPL) to tyrosine. This was demonstrated by nutritional and physiological approaches. The enzymic basis for this unusual ability was shown to be the broad specificity of a particulate, unidirectional, pyridine-nucleotide-independent lactate dehydrogenase. This enzyme, denoted [iLDH], has been implicated in a pathogenic mechanism whereby host-derived lactate is linked to increased gonococcal oxygen consumption and electron transport. A similar role for HPL, a metabolite available in human host tissues, may provide a selective basis to explain evolution of broadened [iLDH] specificity in Neisseria. The interplay between aromatic metabolism and [iLDH] suggests new approaches for manipulating the host-pathogen relationship.
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Iron-repressible Outer-membrane Proteins of Pasteurella haemolytica
More LessThe outer-membrane protein (OMP) profile of Pasteurella haemolytica grown under iron-replete and iron-restricted conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A serotype 1 isolate induced the synthesis of a new 77000 M r OMP in iron-restricted media while two other proteins of 100000 M r and 71000 M r were synthesized in increased amounts. None of these proteins were peptidoglycan-associated or heat-modifiable, and only the 100000 M r protein showed some degree of disulphide cross-linking. Kinetic analysis revealed that the iron-repressible proteins appeared in the outer membrane within 15 min of establishment of iron-restricted conditions. Analysis of P. haemolytica isolates representing serotypes 1 to 12 showed that iron-repressible OMPs of 77000 M r and 71000 M r could be induced in all 12 serotypes but that there was some variability in the expression of the 100000 M r protein. Immunoblotting of OMPs with convalescent sera from P. haemolytica-ifected calves indicated that antibodies directed against all three iron-repressible OMPs were present, suggesting that these proteins were expressed in vivo.
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Characterization of the Outer-membrane Proteins of Haemophilus parainfluenzae Expressed under Iron-sufficient and Iron-restricted Conditions
More LessSDS-PAGE of the outer-membrane (OM) proteins of Haemophilus parainfluenzae P205 grown under iron-sufficient conditions revealed three major proteins of 40, 37 and 13 kDa. In addition, growth under conditions of iron-restriction resulted in the expression of at least four iron-repressible OM proteins (IROMPs) of 72, 81, 88 and 90 kDa. OM proteins of 40 and 13 kDa were non-covalently associated with peptidoglycan and were resistant to digestion with trypsin. A 38 kDa peptidoglycan-associated protein, which was masked by the abundant 37 kDa protein, was also observed following tryptic digestion of whole cells or OMs. Neither the 37 kDa protein (which was heat-modifiable) nor the IROMPs were peptidoglycan-associated, and both were cleaved following treatment of whole cells with trypsin, indicating that they are exposed at the cell surface. A variety of IROMPs from five other H. parainfluenzae strains was also observed. In each strain, both the IROMPs and a major protein of 37 kDa were exposed at the cell surface.
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- Physiology And Growth
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The Effect of Nutrient Limitation on the Competition between an H2-uptake Hydrogenase Positive (Hup+) Recombinant Strain of Azotobacter chroococcum and the Hup− Mutant Parent in Mixed Populations
More LessCompetition studies in continuous culture between a Hup− mutant of Azotobacter chroococcum and its presumed isogenic Hup+ recombinant showed that Hup activity benefited the organism under N2-fixing, sucrose- or phosphate-limiting conditions but was ineffective or disadvantageous under O2-, sulphate- or iron-limitation.
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Requirements for, and Cytoplasmic Concentrations of, Sulphate and Chloride, and Cytoplasmic Volume Spaces in the Halophilic Bacterium Ectothiorhodospira mobilis
More LessEctothiorhodospira mobilis is a halophilic phototrophic bacterium that has been isolated from soda lakes containing high concentrations of sulphate, chloride and carbonates. It utilizes reduced sulphur compounds as photosynthetic electron donors and oxidizes them to sulphate, but can also grow photoheterotrophically with sulphate as sole sulphur source. The requirements for, and the cytoplasmic concentrations of, sulphate and chloride have been determined. High concentrations of sulphate are neither required for nor inhibit growth. Although chloride is by far the dominant anion of the environment, growth of E. mobilis occurs in the absence of added chloride. Sodium chloride can be replaced by sodium sulphate and sodium carbonate. Chloride is excluded from the cytoplasm with decreasing ratios of cytoplasmic/external chloride at increasing external chloride concentrations (under iso-osmotic conditions).
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Reductive and Non-reductive Mechanisms of Iron Assimilation by the Yeast Saccharomyces cerevisiae
E. Lesuisse and P. LabbeIron reduction and uptake was studied in wild-type and haem-deficient strains of Saccharomyces cerevisiae. Haem-deficient strains lacked inducible ferri-reductase activity and were unable to take up iron from different ferric chelates such as Fe(III)-citrate or rhodotorulic acid. In contrast, ferrioxamine B was taken up actively by the mutants as well as by the wild-type strains. At a low extracellular concentration, uptake was insensitive to ferrozine and competitively-inhibited by Ga(III)-desferrioxamine B. Extracellular reductive dissociation of the siderophore occurred at higher extracellular concentrations. Two mechanisms appear to contribute to the uptake of ferrioxamine B by S. cerevisiae: one with high affinity, by which the siderophore is internalized as such and another with lower affinity by which iron is dissociated from the ligand prior to uptake.
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Regulation of the Production of Hemicellulolytic and Cellulolytic Enzymes by a Streptomyces sp. Growing on Lignocellulose
More LessA Streptomyces sp. isolated from compost degraded the hemicellulose fraction of straw efficiently but apparently not native cellulose. Ball-milled straw induced endoglucanase, β-glucosidase, β-xylanase and β-xylosidase. Carboxymethylcellulose, cellotetraose and cellotriose induced cellulolytic enzymes specifically whereas cellobiose acted as inducer for β-glucosidase only. Cellotriose and cellotetraose induced β-glucosidase, but only partially induced endoglucanase. Hemicellulose (in the form of xylan) and xylobiose induced only β-xylanase and β-xylosidase. Kraft lignin and syringic acid induced β-xylanase and endoglucanase but not the other enzymes. 3,4-Dimethoxycinnamic acid slightly induced β-xylanase whereas 3,5-dimethoxy-4-hydroxycinnamic acid specifically induced endoglucanase. Neither veratric acid nor vanillic and ferulic acids induced any of the cellulolytic or hemicellulolytic enzymes. Enzyme production was subject to a form of carbon catabolite repression. Endoglucanase and β-xylanase were excreted into the culture medium. Four protein components, one acidic (pI 5·2) and three basic (pI 8·15, 8·45 and 8·65) exhibited β-xylanase activity. Two acidic components (pI 3·55 and 3·75) displayed endoglucanase activity.
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Phosphate-irrepressible Alkaline Phosphatase of Zymomonas mobilis
More LessThe phosphate regulation and subcellular location of the hydrolytic enzyme alkaline phosphatase were investigated in the Gram-negative bacterium Zymomonas mobilis. The biosynthesis of alkaline phosphatase was not derepressed at a low phosphate concentration as is generally observed in other micro-organisms, nor was it repressed by high phosphate concentrations in the medium. The enzyme level was rather constant during the growth phases in batch culture, at a value at least 8.4-fold lower than that observed in Escherichia coli. The alkaline phosphatase of Z. mobilis was found associated with the membrane fraction after cell disruption, osmotic shock treatment or spheroplast formation. This is a rather unusual location, since most of the alkaline phosphatases from Gram-negative bacteria have been shown to be periplasmic enzymes. Activity staining on polyacrylamide gels after two-dimensional electrophoresis revealed two isoforms of alkaline phosphatase, each of approximate molecular mass 56 kDa. These two forms belong to a group including the more basic envelope proteins of Z. mobilis. Our results indicate that the enzyme from Z. mobilis differs markedly from typical alkaline phosphatases of other Gram-negative bacteria.
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- Plant-Microbe Interactions
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Development of Monoclonal-antibody-ELISA, -DOT-BLOT and -DIP-STICK Immunoassays for Humicola lanuginosa in Rice
More LessMonoclonal antibodies (MAbs) were raised against Humicola lanuginosa, a thermophilic, saprophytic fungus that is commonly isolated from freshly harvested rice grains in Indonesia. Mice were immunized by direct injection into the peritoneum, without prior concentration, of fresh cell-free surface washings from a solid agar slant culture. Hybridoma supernatants were screened by ELISA using wells coated with a dilution of the immunogen. From one fusion 403 hybridoma clones were obtained yielding 52 cell lines secreting antibodies positive for H. lanuginosa. Twelve cell lines were re-cloned, grown in bulk and tested against other storage fungi. Most of the MAbs raised were IgM antibodies that cross-reacted with several of the storage fungi and/or uninfected rice grains. However, the IgM antibody EC6 did not recognize antigens from rice grains and cross-reacted strongly with only one other test fungus, Penicillium variabile, and partially with two others. This MAb was used to develop a highly sensitive DIPSTICK immunoassay to detect the fungus in infected grains. These assays are simple to perform, require no equipment and are suitable for field use by untrained workers.
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Detection of Low-M r Glycoproteins in Surface Washes of Some Fungal Cultures by Gel-filtration HPLC and by Monoclonal Antibodies
More LessSurface washings were prepared from cultures of fungi grown on agar slants and the wash components were fractionated by gel-filtration HPLC. A range of low-M r glycoproteins (< 10000) were detected in all the washes, and in some species several high-M r glycoprotein components (> 150000) were also present. The HPLC analyses indicated species-specific different in the profiles. Regions of specific antigenicity within profiles were detected by monoclonal antibodies.
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Anthoceros-Nostoc Symbiosis: Immunoelectronmicroscopic Localization of Nitrogenase, Glutamine Synthetase, Phycoerythrin and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in the Cyanobiont and the Cultured (Free-living) Isolate Nostoc 7801
More LessLocalization of nitrogenase, glutamine synthetase (GS), phycoerythrin (PE) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was studied with immunocytochemical techniques in the cyanobiont and the free-living cultured isolate Nostoc 7801 of Anthoceros punctatus. In both cases nitrogenase was located in heterocysts only and was uniformly distributed within the cell. GS was located both in heterocysts and vegetative cells, with a uniform cellular distribution in each cell type. Whereas heterocysts of Nostoc 7801 had about twofold higher label than vegetative cells, labelling in heterocysts and vegetative cells of the cyanobiont was similar. While the GS content of the vegetative cells of the cyanobiont and Nostoc 7801 was comparable, the apparent GS content of the cyanobiont heterocysts was 60% less than that in Nostoc 7801 heterocysts. PE and RuBisCO were located in vegetative cells only. PE was located on thylakoid membranes and RuBisCO in the cytoplasm and carboxysomes. In each case the pattern of labelling in the cyanobiont and Nostoc 7801 was similar.
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- Systematics
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Nitrogen Fixation by New Strains of Pseudomonas pseudoflava and Related Bacteria
More LessFive representative bacterial strains resembling Pseudomonas pseudoflava, chosen from among forty strains isolated by three different procedures, were characterized by phenotypic and genotypic analyses. They were shown to belong to the species P. pseudoflava, or to a distinct, but closely related, cluster. In contrast to the initial isolates of this species, most of the new strains were able to fix N2, as shown by their growth in nitrogen-free medium and by the acetylene reduction test. Moreover, DNA regions of these new strains were homologous to Klebsiella pneumomae nifHDK genes, confirming the presence of nitrogenase genes. The nif genes are probably chromosomally located, since no plasmid was detected in these strains. The presence of complete but defective nif genes in P. pseudoflava GA3 (the type strain, which does not fix N2) was excluded, as no homology was found between K. pneumoniae nif genes and P. pseudoflava GA3 total DNA. The isolation conditions seem to determine the selection of P. pseudoflava N2-fixing strains, since selection for H2-chemolithoautotrophs gave no N2-fixing isolates, whereas H2-chemolithoheterotrophic selection conditions (with combined nitrogen) yielded a majority of N2 fixers.
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‘Haemophilus gallinarum’ - a Re-examination
More LessThe growth factor requirements and other properties of two strains of avian haemophili, labelled as ‘Haemophilus gallinarum’(an X- and V-factor-dependent organism) and stored since the 1940s and 1950s, were examined. Both strains were X-factor independent and V-factor dependent and possessed the typical biochemical serological and pathological properties of H. paragallinarum. In experiments repeating the tests used in the 1930s that reported the existence of X- and V-factor-dependent avian haemophili, we found that the methodology used resulted in reference strains of H. paragallinarum (X-factor independent and V-factor dependent) appearing to be X- and V-factor dependent. It is likely that the early descriptions of the aetiological agent of infectious coryza as an X- and V-factor-dependent organism were incorrect.
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