- Volume 136, Issue 10, 1990
Volume 136, Issue 10, 1990
- Sgm Special Lecture
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- Biochemistry
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Purification and properties of an extracellular endoglucanase from Myceliophthora thermophila D-14 (ATCC 48104)
More LessAn extracellular endoglucanase (1,4-β-glucanohydrolase, EC 3.2.1.4) produced by Myceliophthora thermophila D-14 (ATCC 48104) has been purified to homogeneity by ammonium sulphate precipitation and two consecutive ion-exchange chromatographic steps on DEAE-Sephadex A-50 columns. The enzyme was purified 13·8-fold and was homogeneous by analytical PAGE and SDS-PAGE. It has a high apparent M r, of about 100000. The pH and temperature optima for its activity were 4·8 and 65°C respectively. The K m of the purified enzyme for CMC (sodium salt) was 3 mg ml−1. The enzyme displayed low activity toward salicin and p-nitrophenyl β-d-glucoside. The activity was enhanced in the presence of Na+, K+ and Ca2+ but effectively inhibited by Hg2+, Fe2+, Mg2+, Cu2+ and NH4 +. Inhibition studies indicated that the enzyme may be a metalloprotein and/or that it requires metal lons for its optimum activity.
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Purification and characterization of alkaline endo-1,4-β-glucanases from alkalophilic Bacillus sp. KSM-635
Two carboxymethylcellulases (CMCase, 1,4-1,4-β-d-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0·5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9·5, and their optimum temperatures were around 40 °C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-d-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-d-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.
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Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum
More LessThe rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 104 g-min (fraction P1) and 105 g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0·4–0·6 μm in diameter and which had a mean equilibrium density of 1·22–1·24 g ml−1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 × 106 g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).
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Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite
More LessThe proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7·4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0·5 mm-nitrite with 10 mm-ascorbate stopped growth completely. In partially-purified preparations 4·1 mm-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78 %) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO)2(SR)2] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3·6 mm-NaNO2 and 3·6 mm-ascorbate. It is concluded that the effects of nitrite on pre-formed ironsulphur proteins are not convincing as a basis for the lethal effects on bacterial cells.
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Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes
More LessCertain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl ([Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0·72 mm) with iron (1·44 mm) and cysteine (2·16 mm) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 µm-nitrite. If added to the initial culture medium, it prevented growth at 5 µm-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the ‘Perigo factor’, obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin’s black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 µm-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 µm-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2·5 µm-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2·03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1·65. There was no correlation between growth inhibition and the g = 2·03 signal, though there was a better correlation between inhibition and the g = 1·65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mm-cysteine, 3·6 mm-FeSO4 and 0·72 mm-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.
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- Development And Structure
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Switching at the cellular level in the whiteopaque transition of Candida albicans
More LessThe ‘white-opaque transition’ in Candida albicans strain WO-1 provides a unique system for analysing high-frequency switching at the cellular level because of the difference in the budding phenotypes of the white and opaque phases. Single white and opaque cells were placed on agar and monitored for the dynamics of cell division, microcolony genesis and switching to the alternative phase. It is demonstrated that at 24 °C, opaque cells can switch directly to white cells but white cells first generate an elongate, pseudohyphal-shaped precursor in the transition to an opaque cell. Cells in either phase can generate a daughter cell in the alternative phase, then revert immediately to the genesis of subsequent daughter cells in the original phase. By developing a mathematical model for switching at the cellular level which subtracts mother cells and switched daughter cells from the pool of switching candidates, the probability for an opaque cell to generate a white daughter cell in any single generation was calculated to be 1·0 × 10−1, and the probability for a white cell to generate an opaque daughter cell in any single generation was calculated to be 1·7 × 10−5 at 24 °C on nutrient agar. The mean number of generations before an opaque cell generated a white daughter cell was calculated to be 3·4 and the mean number before a white cell formed an opaque cell was calculated to be 15·8 at 24 °C on nutrient agar. Finally, high-temperature induction of the opaque to white transition was analysed at the cellular level and demonstrated to involve frequent bipolar formation of white daughter cells on the original opaque mother cell, and in some cases intermediate phenotypes.
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- Genetics And Molecular Biology
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Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F
More LessA hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.
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Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46
More LessGenomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into λgt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2·3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley-glucan, Avicel, filter paper and p-nitrophenyl-d-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.
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Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme
More LessThe nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.
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Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation
The Escherichia coli–Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 106 transformants per μg of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction–modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction–modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.
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Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2
More LessThermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 °C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 °C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.
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- Pathogenicity And Medical Microbiology
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Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans
More LessDifferences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.
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Invvestigation of the structure and localizatioin of the urease of Helicobacter pylori using monoclonal antibodies
More LessThe urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypetides (61 kDa and 28 kDa) were observed in urese-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypetptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for anzyme actiity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacterial and ultrathin cryosectioned bacterial the enzyme was located on the cell surface and in material apparently shed from that surface.
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Subcellular localization of Mycobacterium leprae-specific phenolic glycolipid (PGL-I) antigen in human leprosy lesions and in M. leprae isolated from armadillo liver
More LessPhenolic glycolipid (PGL-I), an antigen specific to Mycobacterium leprae, was localized subcellularly in M. leprae residing in human skin, in M. leprae isolated from armadillo liver (‘isolated M. leprae’) and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. Ultracryosections and Araldite sections of comparable specimens were used for comparison of localization results. A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathin sections. K4M-immunogold methods gave very satisfactory quantitive gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosections and the quantitatively superior Araldite sections, but new sites were also localized. Cell walls in human M. leprae and in isolated M. leprae possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated M. leprae cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprae-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. leprae, and (2) intra- and extracellularly in epidermal areas where basal cells harboured M. leprae in untreated multibacillary patients.
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Isolation of recombinant fragments of the major outer-membrane protein of Chlamydia trachomatis: their potential as subunit vaccines
More LessRecombinant fragments of the major outer-membrane protein (MOMP) of Chlamydia trachomatis, expressed at high levels in Escherichia coli, were isolated and purified. Antisera to the recombinant proteins reacted preferentially with overlapping synthetic peptides covering the immunoaccessible variable segments of MOMP. These sera also reacted in a species-specific manner with the surface of intact infectious elementary bodies, and in a Chlamydia genus-specific manner in assays using denatured or bound chlamydial antigens. The ability of recombinant MOMP preparations to elicit antibody to the surface of chlamydial elementary bodies raises the possibility that these proteins may be useful for chlamydial vaccine development.
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- Physiology And Growth
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Inhibition of the dimorphic transition of Candida albicans by the ornithine decarboxylase inhibitor 1,4-diaminobutanone: alterations in the glycoprotein composition of the cell wall
More LessHyphal development in Candida albicans was selectively blocked by the ornithine decarboxylase competitive inhibitor 1,4-diaminobutanone (DAB). Inhibition of hyphal development required DAB during both yeast inoculum growth and subsequent incubation at 37°C to induce mycelial growth. This effect was not due to general growth inhibition since DAB did not inhibit yeast growth, and reduced protein synthesis by 30% at most. Moreover, protein synthesis was unaffected by DAB when cells were pre-grown in drug-containing media. Since DAB inhibited dimorphic transition at 37°C, morphology-and temperature-dependent protein synthesis could be distinguished. DAB stimulated the synthesis of several yeast wall-proteins, irrespective of morphology or growth temperature, and two at 37°C only, but it inhibited the synthesis of a single mycelial-specific glycoprotein species.
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Lactate and ethanol dehydrogenase activities in continuous cultures of Clostridium thermosaccharolyticum LMG 6564
More LessThe pattern of ethanol and lactate formation by continuous cultures of Clostridium thermosaccharolyticum LMG 6564 under glucose limitation is affected by culture conditions such as pH and dilution rate. NADH- and NADPH-mediated lactate dehydrogenase (LDH; EC 1.1.1.27) and alcohol (ethanol) dehydrogenase (ADH; EC 1.1.1.1 and EC 1.1.1.2) activities were measured in cell extracts from continuous cultures grown under different conditions. In conditions of high product formation, the NADH-mediated reaction was higher than the NADPH-mediated reaction for both LDH and ADH. LDH showed an absolute requirement for fructose 1,6-bisphosphate (FBP). Both NADH- and NADPH-linked LDH reactions were cytoplasmic, not sensitive to oxygen, and had a pH optimum of 6·0–6·5; the temperature optimum was 55–60 °C. The reverse reaction (lactate oxidation) could not be demonstrated. ADH activity was found in the particulate fraction of the cell lysate and was sensitive (not completely, but irreversibly) to oxygen. The temperature and pH optima were 43 °C, pH 7·0 and 45 °C, pH 8·8 for the NADH- and NADPH-mediated reactions, respectively. The production of at least two different ADHs is likely. LDH and ADH seemed to be regulated at the level of enzyme synthesis (direct correlation between the in vitro activities and the lactate and ethanol yields in the culture) with a second regulation of LDH by FBP at the reaction level.
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Oxygen toxicity, respiration and behavioural responses to oxygen in free-living anaerobic ciliates
More LessThree species of marine, anaerobic free-living ciliates (Metopus contortus, Plagiopyla frontata and Parablepharisma collare) were studied with respect to their relation to O2. Survival was adversely affected at O2 tensions exceeding 1–2% of atmospheric (air) saturation (atm. sat.): Parablepharisma collare survived for about 1 h at 100% atm. sat., while the other two species survived the treatment for up to 2 d. Survival in the presence of O2 depended on the composition of the medium; cells survived longer in clean seawater than in culture medium which, when exposed to O2, became toxic. This is probably related to peroxide production mediated by solutes in the culture medium: addition of catalase prolonged survival. The ciliates did not contain catalase. Two of the species harbour endosymbiotic methanogens; at O2 tensions exceeding 2% atm. sat. the bacteria were inactivated, but they remained viable even after more than 5 h exposure to atmospheric O2 tension. Metopus contortus and Plagiopyla frontata respired O2 at rates similar to those of aerobic species; this O2 uptake is not coupled to energy conservation since the ciliates do not contain cytochromes, and low O2 tensions do not stimulate growth. O2 consumption is probably a detoxification mechanism which can maintain an intracellular anaerobic environment at a low ambient O2 tension. Metopus contortus and Plagiopyla frontata showed chemosensory behaviour in response to O2; this allowed them to find and remain within anaerobic microhabitats.
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Isolation and characterization of a Rhodotorula glutinis mutant defective in glucose transport
More LessMutants of the obligatorily aerobic yeast Rhodotorula glutinis, obtained by treating wild-type cells with N-methyl-N′-nitro-N-nitrosoguanidine, were selected on fructose medium containing 1% 2-deoxy-d-glucose. One mutant, designated R33, displayed a significantly decreased initial rate of uptake of d-glucose, 2-deoxy-d-glucose, d-galactose and d-xylose, and was unable to accumulate the latter three sugars. However, the residual monosaccharide transport was still energy-dependent. The mutant displayed significantly altered transport parameters for d-glucose and d-xylose. These results are evidence for the isolation of a mutant defective in the glucose carrier.
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Lipid and fatty acid composition of freshwater cyanobacteria
More LessFour species of freshwater cyanobacteria (Anabaena cylindrica, Anacystis nidulans, Nostoc canina and Nostoc muscorum) contained as major lipid classes monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols and phosphatidylglycerols. Unlike photosynthetic eukaryotes, cyanobacteria incubated for 7 d in the dark suffered no decrease in the concentrations of these classes, except for N. muscorum. Growth, photosynthesis and nitrogen fixation were 30–40 % lower after dark incubation. The nitrogen-fixing cyanobacteria, Anabaena cylindrica, N. canina and N. muscorum, contained alcohol glycosides and a highly-polar unknown glycolipid at high concentrations. The proportion of these two lipid classes decreased in the dark in N. muscorum alone. Extracts from Anacystis nidulans and N. canina, and to a lesser extent N. muscorum, contained sterols, whose concentration increased after dark incubation. Anabaena cylindrica contained considerable concentrations of linolenic acid in its total lipid, which did not decrease on dark incubation, and was not present mainly in monogalactosyldiacylglycerols as in photosynthetic eukaryotes. Palmitoleic acid, which is primarily confined to phosphatidylglycerols in photosynthetic eukaryotes, was distributed among the major lipid classes of N. canina.
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Is cyanophycin involved in the integration of nitrogen and carbon metabolism in the cyanobacteria Anabaena cylindrica and Gleothece grown on light/dark cycles?
More LessIn Anabaena cylindrica, protein synthesis continued during dark periods at 80 % of the rate observed in the light. Since nitrogen fixation ceases in the dark, this implies that fixed nitrogen accumulates during the light periods to supply amino acids for protein synthesis in the dark. Measurements of cyanophycin and the distribution of nitrogen in subcellular fractions indicated that cyanophycin does not represent a significant proportion of total cell nitrogen, nor does its concentration vary across the light/dark cycle. Similar results were obtained with Gloeothece; there was no evidence that cyanophycin accumulated during the dark to support protein synthesis in light. Cyanophycin does not, therefore, appear to serve as a dynamic temporary storage form of newly fixed nitrogen in the integration of light and dark metabolism. We also investigated an immunological approach to the measurement of cyanophycin and its turnover. Cyanophycin is immunogenic, and in the pure state could be assayed by radioimmunoassay, which had greater sensitivity than traditional assay procedures. The insolubility of cyanophycin at neutral pH, however, prevented the successful development of methods for quantifying cyanophycin turnover in cell extracts.
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Transient accumulations of cyanophycin in Anabaena cylindrica and Synechocystis 6308
More LessCyanophycin, the nitrogen reserve compound in cyanobacteria, has a dynamic metabolism during transitions between the metabolic states of nitrogen deficiency and nitrogen repletion and vice versa. Cyanophycin was transiently synthesized when ammonia-grown Anabaena cylindrica or Synechocystis 6308 consumed a limited amount of ammonia. It was synthesized during the phase of declining ammonia concentration, and its degradation was complete by the time the external ammonia had been completely consumed. When nitrogen-starved cells of A. cylindrica or Synechocystis 6308 were given a usable source of fixed nitrogen, cyanophycin again accumulated transiently. This synthesis of cyanophycin was triggered by the suddenly renewed availability of fixed nitrogen, and not by the subsequent decline in the external concentration of fixed nitrogen. The cyanophycin was degraded when balanced exponential growth was again possible. Transient accumulations of cyanophycin with similar kinetics were also observed when ammonia was added to nitrogen-fixing A. cylindrica. It is suggested that cyanophycin serves as a dynamic reservoir which separates the environmental supply of fixed nitrogen from the metabolic demands of the cells, and that this provides a mechanism which enables cyanobacteria to maximize their share of any available fixed nitrogen. This would give cyanobacteria a competitive advantage over other organisms.
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- Plant-Microbe Interactions
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Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions
More LessPrevious studies with labelled N and C have indicated synthesis and accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions similar to those found in soybean nodules. Low 2-oxoglutarate dehydrogenase (OGDH) activity might have accounted for this observation, but similar levels of enzyme activity were found in bacteroids isolated anaerobically or aerobically and in cultured bacteria. However, OGDH from B. japonicum bacteroids was strongly inhibited by NADH, and the degree of inhibition depended on the NADH:NAD ratio. Determination of endogenous levels of NAD and NADH gave NADH:NAD ratios of 0·19 and 0·83 in bacteroids isolated under aerobic and anaerobic conditions, respectively. A ratio of 0·83 resulted in more than 50 % inhibition of OGDH in vitro, and this would be consistent with channelling of 2-oxoglutarate to glutamate. [14C]Glutamate supplied to bacteroids was metabolized to CO2 slowly relative to the respiration of malate, and essentially no labelling of products of glutamate metabolism such as arginine, proline, glutamine and 4-aminobutyrate (GAB) was found. Attempts to trap 14C in GAB by supplying unlabelled GAB or transaminase inhibitors with [14C]glutamate were unsuccessful. The finding that glutamate decarboxylase was essentially absent in six different strains of B. japonicum was consistent with the labelling results and indicated that conversion of glutamate to succinate via GAB is slow or nil. The inhibition of OGDH by a high NADH:NAD ratio and the absence of the GAB shunt are complementary mechanisms which probably account for the accumulation of glutamate.
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- Systematics
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Demonstration of synonymy between the plant pathogens Pseudomonas avenae and Pseudomonas rubrilineans
More LessStrains of Pseudomonas avenae Manns 1909 and Pseudomonas rubrilineans ( Lee et al., 1925 ) Stapp 1928 were compared using physiological, biochemical and serological tests together with an examination of plant host range. Minor differences were found between the physiological and biochemical characteristics of the two pathogens, while no differences were detected in pathogenicity, host range, cellular protein profiles, direct fluorescent straining, and dot-immunobinding assays. P. avenae and P. rubrilineans were distinctly different from other non-fluorescent pseudomonads. We were unable to differentiate P. avenae strains from those of P. rubrilineans, and we propose that they be regarded as a single species, retaining the name Pseudomonas avenae.
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The determination of ubiquinone profiles by reversed-phase high-performance thin-layer chromatography as an aid to the speciation of Legionellaceae
More LessUbiquinones extracted from 24 strains of Legionella pneumophila (including the type strains of serogroups 1–14 and one proposed new serogroup) and from 44 strains of other Legionella species (including 28 type strains and strains of five proposed new species) were analysed by reversed-phase thin-layer chromatography. Ubiquinone profiles as determined by this method were reproducible, both qualitatively and semi-quantitatively, and provided information to aid in the identification of species of Legionella. Results of ubiquinone profiles determined by different laboratories were compared. Some quantitative differences in results between laboratories were observed, which may be due to different analytical procedures. For this reason laboratories should establish their own library of ubiquinone profiles. New information is presented on the ubiquinone profiles of seven Legionella species: L. birminghamensis, L. brunensis, L. cincinnatiensis, L. moravica, L. quinlivanii, L. tucsonensis and proposed new species no. 1347, ‘L. worsleiensis’
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Sensitivities of various Oomycetes to hymexazol and metalaxyl
More LessThe effect of hymexazol on the linear extension of hyphae of a range of Oomycetes was examined. Twenty-five species were compared using four fungicide concentrations. Cluster analysis was used to look for similarities between species. The response of each taxon to the different concentrations of hymexazol was modelled. The parameters of these curves were then subjected to multivariate analysis. Both analyses revealed close relationships between the sensitivity to the fungicide and the current classification of the Oomycetes. Comparisons were made with the effect of metalaxyl on the same isolates.
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Differentiation between Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus paraphrophilus by multilocus enzyme electrophoresis
More LessGenetic relationships among isolates assigned to Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and H. paraphrophilus were determined by analysis of electrophoretically demonstrable allelic variation in 14 structural genes encoding metabolic enzymes. Among the 51 isolates analysed there were 25 electrophoretic types (ETs), among which mean genetic diversity per locus was 0·753. Cluster analysis of ETs demonstrated one well-defined group of 11 ETs representing solely the genotypes of all 17 isolates assigned to A. actinomycetemcomitans. The remaining 14 ETs represented the genotypes of the 34 isolates of H. aphrophilus and H. paraphrophilus. With the exception of ATCC 13252, all strains of H. aphrophilus were closely related, whereas strains assigned to H. paraphrophilus included distantly related lineages, some of which were similar to those of H. aphrophilus and should be assigned to this species. Thus, the study showed that there is no significant overall genetic similarity between A. actinomycetemcomitans and the two Haemophilus spp.
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