- Volume 130, Issue 11, 1984
Volume 130, Issue 11, 1984
- Sgm Special Lecture
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- Biochemistry
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Protein Degradation in Extracts of Exponential and Stationary Phase Vibrio Cells
N. G. Car and D. R. WoodsThe degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 °C. Maximum stimulation was obtained with 3 mm-ATP and optimum degradation was at pH 8·0–8·5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.
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An Electrophoretic Analysis of Superoxide Dismutase in Campylobacter spp
More LessSuperoxide dismutase (SOD, superoxide: superoxide reductase, EC 1.15.1.1) activity was studied in 23 strains of Campylobacter spp. using disc polyacrylamide gel electrophoresis. Different enzyme patterns were observed with extracts of different species of Campylobacter; three migration bands were found in all strains of C. sputorum subsp. sputorum and C. sputorum subsp. bubulus (relative mobilities, Rm, 0·57, 0·76 and 0·85), and C. fetus subsp. fetus (Rm 0·60, 0·72 and 0·81), while four migration bands (Rm 0·52, 0·57, 0·73 and 0·82) were found in C. fetus subsp. venerealis. One band (Rm 0.73) was found in C. coli CIP 7080 and two bands (Rm 0·59, 0·73) in C. jejuni CIP 702. Superoxide dismutase activities were very high in the Campylobacter strains, especially in C. fetus subsp. fetus [specific activity 7·8–55·7 U (mg protein)−1] compared with those in Escherichia coli (1·5 U mg−1), Propionibacterium acnes (1·6 U mg−1) and Veillonella alcalescens (0·2 U mg−1).
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Respiratory Adaptation of Anaerobically Grown Saccharomyces uvarum: Changes in Distribution of Enzymes
More LessSubcellular fractionations using metrizamide density gradients revealed intermediary stages of respiratory adaptation of Saccharomyces uvarum grown anaerobically in the presence of the density label 16-bromo-9-hexadecenoic acid. Prior to adaptation, activities of malate dehydrogenase and oligomycin-sensitive ATPase were contained within a membrane population at ρ = 1·20 g ml−1. After 10 min adaptation cytochrome c oxidase activity was associated with these membranes, with ATPase-containing membranes at lower densities (ρ = 1·05 to 1·14 g ml−1) and with membranes containing malate dehydrogenase at higher density (ρ = 1·24 g ml−1). After further adaptation these enzymes were associated firstly with two distinct membrane populations at ρ = 1·17 and 1·20 g ml−1 and finally with a single population of mitochondria at ρ = 1·16 g ml−1. The significance of these changes is discussed in terms of mitochondrial differentiation. Peroxisomes were evident even in early stages of respiratory adaptation and were well separated from mitochondria in later stages.
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An Enzyme in the Pectinolytic Pathway of Erwinia chrysanthemi: 2-Keto-3-deoxygluconate Oxidoreductase
More Less2-Keto-3-deoxygluconate : NAD 5-oxidoreductase (EC 1.1.1.127) is an enzyme used in pectinolysis by some phytopathogenic bacteria such as Pseudomonas and Erwinia. It converts 2,5-diketo-3-deoxygluconate (DKII) into 2-keto-3-deoxygluconate (KDG) with a concomitant oxidation of NADH. The reaction is reversible in the presence of NAD. The oxidoreductase isolated from Erwinia chrysanthemi was purified 40-fold by protamine treatment, (NH4)2SO4 fractionation, chromatography on DEAE-Sephadex and filtration on Sephadex G-200. The enzyme appeared relatively stable. There was no effect of EDTA or of added ions except Ag+. This ion and the thiol reagent p-chloromercuribenzoate inhibited the enzyme. Other uronic acids, sugars and organic acids tested were neither substrates nor inhibitors of its activity. The optimum pH was 10 for the oxidation of KDG and 7·5 for the reduction of DKII. Both oxidation and reduction reactions showed saturation kinetics with apparent K m values of 7·7 10−3 m for KDG and 4 10−4 m for NAD (at 37 °C, pH 8·6), and of 3·4 10−4 M for DKII and 0·3 10− m for NADH (at 37 °C and pH 7·0). The enzyme reaction was strongly inhibited by NADH at concentrations higher than 2 × 10−4 m.
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Location of the Catalytic Site of the Respiratory Fumarate Reductase of Escherichia coli
More LessThe location of the catalytic site of the membrane-bound respiratory fumarate reductase of Escherichia coli was investigated using mutants and inhibitors of dicarboxylic acid transport. Comparison of apparent K m and V max values for fumarate in intact cells and in inverted membrane vesicles showed that externally added fumarate was required to be transported across the cytoplasmic membrane prior to reduction. The catalytic site of fumarate reductase must therefore be located on the cytoplasmic face of the membrane.
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Further Characterization of Cytochrome P-460 in Nitrosomonas europaea
More LessReduction of cells or extracts of Nitrosomonas europaea with dithionite leads to a peak in absorption spectra at 460 nm. Most of this chromophore is bound to hydroxylamine oxidase, but a small fraction exists as a separate protein, namely cytochrome P-460. An improved purification of both these proteins is described. The mobility of cytochrome P-460 on gel electrophoresis in the presence of SDS corresponded to a molecular weight of 18000, whereas a molecular weight of 52000 was determined by gel filtration. The band obtained by electrophoresis gave a yellow-green fluorescence in UV light. Electrophoresis in the presence of 8 m-urea, or after acid-treatment, resulted in red fluorescence from cytochrome P-460.
We previously reported that extracts from N. europaea gave eight fluorescence bands in SDS-polyacrylamide gels; cytochrome P-460 was found to correspond to the previously unassigned band VI. Electrophoresis of purified hydroxylamine oxidase did not give a band in common with cytochrome P-460. Highly purified cytochrome P-460 gave no detectable flavin-type fluorescence. There was no antigenic similarity between hydroxylamine oxidase and cytochrome P-460.
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- Biotechnology
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Screening for Lignin-degrading Actinomycetes and Characterization of their Activity against [14C]Lignin-labelled Wheat Lignocellulose
More LessA sedimentation chamber and Andersen sampler were used to isolate a range of actinomycetes on selective and non-selective media. The occurrence of different actinomycete groups in natural substrates was compared and strains were screened for the ability to degrade ball-milled straw or to grow on lignin-related phenolic compounds. Evidence for ligninolytic activity in representatives of several genera was obtained by assaying 14CO2 evolution from [14C]lignin-labelled wheat lignocellulose. Most of the straw-degrading isolates were assigned to the genera Thermomonospora and Micromonospora, but only representatives of the latter were found to be active against [14C]lignin. Of the non-straw-degrading strains also examined, some which could utilize phenolic substrates produced 14CO2 from the [lignin-14C]lignocellulose, and two of these were selected for further study. These strains, Thermomonospora mesophila and a Streptomyces sp., attacked [14C]lignin yielding 14CO2 and water-soluble 14C-labelled compounds during primary growth. This activity was not accounted for by the utilization of phenolic acids linked to the carbohydrate fraction of wheat lignocellulose and was unaffected by cultural parameters known to influence lignin degradation by white-rot fungi.
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- Development And Structure
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Chemical Studies of Partially Hydrolysed Lipopolysaccharides from Four Strains of Campylobacter jejuni and Two Strains of Campylobacter coli
More LessLipopolysaccharides (LPS) from four strains of Campylobacter jejuni and two strains of C. coli were partially hydrolysed with 1% acetic acid. Subsequent chloroform extraction led to the formation of a polysaccharide-containing aqueous layer, an interfacial material and a lipid A-containing chloroform layer. The polysaccharides contained the neutral sugars, amino sugars, 2-keto-3-deoxy-octonic acid, and part of the phosphorus present in the undegraded LPS. The lipid As were made up of glucosamine, phosphorus, ester- and amide-linked 3-hydroxytetra-decanoic acid, and ester-linked n-tetradecanoic and n-hexadecanoic acid. The interfacial material was made up of lipid A and undegraded LPS.
When chromatographed on Bio-Gel P-60, the degraded polysaccharides were eluted as two incompletely separated peaks (strains NCTC 11168, NCTC 11351, 11041 and 11101) or as one peak (strains NCTC 11392 and E 8035). All peaks appeared close to the total volume of the column. When the different fractions were re-chromatographed on Bio-Gel P-10, the peaks still appeared close to the total volume of the column. These findings indicate that LPS from C. jejuni and C. coli are devoid of long O-antigenic side-chains.
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Septal Sealing in the Basidiomycete Coriolus versicolor
More LessThe way in which the dolipore apparatus contains hyphal damage, and the process of septal sealing have been studied in Coriolus versicolor using combined light and electron microscopy. The technique used allows the structure of septa in adjacent damaged and undamaged hyphae to be compared. The results show that septal sealing, following damage, is a two stage process. The first is the instantaneous plugging of the pore channel with electron-dense material. The second, beginning several minutes later, involves the detachment of the septal apparatus present in the ruptured compartment and a re-modelling of the septal swelling on the other side of the wall to give a permanent seal. The parenthesomes play no part in the plugging response.
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Re-examination of Capsule Development and Lectin-binding Sites on Rhizobium japonicum 3I1B110 by the Glutaraldehyde/Ruthenium Red/Uranyl Acetate Staining Method
More LessCapsular development and soybean lectin binding properties of Rhizobium japonicum 3I1B110 were examined by the glutaraldehyde/ruthenium red/uranyl acetate method permitting observation of whole bacteria and their polar capsules by transmission electron microscopy. Ultrastructurally, the polar capsule consisted of (i) an electron-dense aggregated material in close contact with the cell surface which stained with ruthenium red and intensively bound soybean lectin, and (ii) a less electron-dense fibrillar material at the periphery of the capsule which stained with ruthenium red but did not bind soybean lectin. The latter material interconnected with the aggregated capsular material at the cell surface. As the culture advanced into stationary phase, these acidic capsular materials were shed into the culture medium where they kept their respective lectin-binding activity. The cells correspondingly decreased in their lectin-binding activity and encapsulation with culture age. However, the remaining encapsulated cells in late stationary phase were still able to bind the lectin in a hapten-specific way.
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- Ecology
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Simulation of Microbial Processes in Estuarine Sediments using Gel-stabilized Systems
More LessGel-stabilized model systems have been developed which simulate the physico-chemical gradients found in estuarine sediments of the River Tay, Scotland. Depth profiles of E h and pH, and of concentrations of NH+ 4, NO− 2 and NO− 2 and dissolved O2 which developed within the gels were similar to those measured in situ in the sediments. The spatial distribution of populations of nitrifying, nitrate respiring and sulphate reducing bacteria in mature gels showed a good correlation with those recorded in the surface sediments of Kingoodie Bay in the Tay estuary. These data show that NH+ 4 oxidation by autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) plays a significant role in the development of pH, NH+ 4, NO− 2 and NO− 3 gradients within the gels, indicating that similar processes may also operate in situ in these sediments.
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Changes in the Nitrate-reducing Community of an Anaerobic Saltmarsh Sediment in Response to Seasonal Selection by Temperature
More LessMeasurement of the nitrate-reducing potential by bacteria in saltmarsh sediment, using a thermal gradient block incubator, revealed seasonal physiological changes in the community. A mesophilic part of the nitrate-reducing community was always present, although it achieved maximum development at the end of the summer and minimum development at the end of the winter. In contrast, a distinct psychrotrophic part of the community achieved maximum development at the end of the winter but disappeared during summer. Chemostat enrichment of nitrate-reducing bacteria at 10 °C isolated predominantly Pseudomonas spp., but Vibrio spp. predominated in enrichments at 25 °C. The observed seasonal changes in situ might reflect differential seasonal selection of these two groups of bacteria.
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- Genetics And Molecular Biology
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NAD Metabolism In Salmonella typhimurium: Isolation of Pyridine Analogue supersensitive (pas) and pas suppressor Mutants
More LessMutants of Salmonella typhimurium supersensitive to the nicotinic acid analogue 6-aminonicotinic acid (6ANA) were isolated as unable to grow on what are normally subinhibitory concentrations of the analogue. The mutations were classified on the basis of their map positions as pasA (89–92 units), pasB (66–69 units), pasC (18–22 units), pasD (18 units) and pasE (55 units). The mutants exhibited a wide range of minimal inhibitory concentrations towards 6ANA, and several were affected in terms of growth. The data suggest that most of the mutations probably reside in genes whose products utilize nicotinamide adenine dinucleotide (NAD) as a cofactor, the altered gene products being more sensitive to internal 6-amino NAD concentrations. Secondary mutations which suppress the Pas− phenotype were found to reside in the following NAD-related loci; pncB, nadB and nadD. Two of the pncB mutants appear to be affected in the expression of NAPRTase while several of the nadB mutants are apparently insensitive to feedback inhibition by internal NAD concentrations.
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Resistance to Inhibitors of RNA Polymerase in Actinomycetes Which Produce Them
More LessResistance to the endogenous antibiotic was studied in three actinomycetes that produce inhibitors of RNA polymerase. The three producers, Nocardia mediterranei (rifamycin producer), Streptomyces spectabilis (streptovaricin producer) and Streptomyces lydicus (streptolydigin producer), were each highly resistant to the antibiotic they produce (MIC >200 μg ml−1) and in vivo RNA synthesis was also resistant. However, cross-resistance to the other RNA polymerase inhibitors was not found. Resistance to these antibiotics was due to target site modification, since the RNA polymerase enzymes of the three producing organisms were highly resistant in vitro to the corresponding antibiotic, and no antibiotic-inactivating enzymes were detected. A mutant was isolated from S. spectabilis which was sensitive to steptovaricin (its own product) and also showed an increased sensitivity to rifamycin and streptolydigin. This mutant had RNA polymerase which was extremely sensitive to the three antibiotics.
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Isolation, Characterization and Mapping of Mandelate Pathway Mutants of Acinetobacter calcoaceticus
More LessMutants of Acinetobacter calcoaceticus EBF 65/65 that could not grow on intermediates of the mandelate or benzyl alcohol pathways were isolated and in some cases the enzymic lesions were identified. Several catabolic markers were mapped using the plasmid pAVl. The mandelate genes appeared to be clustered near the auxotrophic marker phe-1 but were not all contiguous with each other. The gene responsible for the appearance of the novel l(+)-mandelate dehydrogenase appeared to be close to a gene responsible for the activity of the original d(−)-mandelate dehydrogenase.
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Mitochondrial DNA in Fusarium oxysporum is a 46·5 Kilobase Pair Circular Molecule
More LessPurified mitochondria were obtained from the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici by mechanical disruption of protoplasts, followed by differential and density gradient centrifugation. DNA, extracted from the mitochondria, was shown by electron microscopy and restriction endonuclease analysis to be a 46·5 kilobase pair circular molecule.
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Characterization of the Fumarase Gene of Bacillus subtilis 168 Cloned and Expressed in Escherichia coli K12
More LessThe fumarase (citG) gene of Bacillus subtilis 168 has been identified in a collection of λ phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA have been subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement Escherichia coli and B. subtilis fumarase mutations has been examined. Two EcoRI fragments of 1·5 and 5·1 kb are both required for fumarase expression in E. coli and B. subtilis. The level of fumarase activity from a single copy of the B. subtilis citG gene expressed in E. coli is approximately one-tenth of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene has been located within the cloned DNA by transposon mutagenesis and by expression studies, which have also identified a polypeptide of M r 49000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide have indicated the direction of transcription of the citG gene.
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Characterization and Physical Analysis of a 3,5-Xylenol Degradative Plasmid in Pseudomonas putida
More LessPseudomonas putida NCIB 9869 carries a transmissible plasmid pRA500 of approximately 500 kb which encodes the degradation of 3,5-xylenol via a gentisate pathway. Several mutant strains which were unable to utilize 3,5-xylenol were isolated and these strains either carried deleted derivatives of pRA500 or lacked plasmid DNA. Biochemical and restriction endonuclease analysis of the wild-type and mutant strains showed that the structural and/or regulatory genes for 3,5-xylenol metabolism were encoded within a 130–140 kb region of pRA500 and that, with the exception of the first enzyme of the pathway, 3,5-xylenol methylhydroxylase, all the enzymes were encoded within a 50–70 kb segment of that region. pRA500 also encoded for resistance to inorganic mercuric ions; the genes for this phenotype were located separately from those for the degradation of 3,5-xylenol.
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Plasmid Involvement in Production of and Immunity to the Staphylococcin-like Peptide Pep 5
More LessThe staphylococcin-like peptide Pep 5 is produced by the penicillin resistant strain Staphylococcus epidermidis 5. This strain is immune to the peptide. Plasmid analysis of S. epidermidis 5 by agarose gel electrophoresis and electron microscopy demonstrated five plasmids with molecular weights ranging from 5·8 × 106 to 29 × 106. Variants of S. epidermidis 5 not producing Pep 5 or which had become penicillin sensitive were induced by various curing treatments. Strains lacking the 13·9 × 106 mol. wt plasmid (pED502) had lost penicillin resistance, and those lacking the 12·3 × 106 mol. wt plasmid (pED503) failed to produce Pep 5. pED503 is also responsible for the immunity of the producer cell to Pep 5. Plasmid pED502 could be transformed into S. aureus RN 981 which then became resistant to penicillin. pED503 could not be transformed into S. aureus RN 981, but could be transformed into S. epidermidis 5 variants previously cured of this plasmid; the transformants then regained the properties of Pep 5 production and immunity.
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- Pathogenicity And Medical Microbiology
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Fractionation of Guinea Pig Serum for an Inducer of Gonococcal Resistance to Killing by Human Serum: Active Fractions Containing Glucopeptides Similar to Those from Human Red Blood Cells
More LessThe resistance of gonococci to complement-mediated killing by serum is important in the pathogenesis of gonorrhoea. Most urethal strains lose this resistance on subculture. The host product(s) which induces the resistance in vivo is therefore fundamental to pathogenesis. Human genital secretions and some sera induced gonococci to serum resistance in vitro. Guinea pig serum was more active than human serum and low molecular weight fractions from it conferred resistance to gonococci in 3 h at 37 °C. Similar active fractions were obtained from human sera. Now guinea pig serum has been further fractionated for the low molecular weight inducer by membrane filtration, gel filtration on Sephadex G25, high performance liquid chromatography (HPLC) with a Spherisorb ODS reverse phase column, chromatography on Sephadex LH20 and HPLC with a Partisil SCX cation exchange column. The small yield (less than 1 mg from 400 ml serum) of highly active material was contaminated with breakdown products from the Partisil SCX column and a mixture of compounds. However, analysis indicated the presence of one or more small glucopeptides containing cysteine, glutamic acid, aspartic acid, threonine, serine, glycine, alanine, valine and lysine. Similar glucopeptides are liberated from fresh human red blood cells in slightly hypertonic saline and samples of them induced gonococci to serum resistance.
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Red Blood Cells, a Source of Factors which Induce Neisseria gonorrhoeae to Resistance to Complement-mediated Killing by Human Serum
More LessLysates of guinea pig or human red blood cells (RBC) contain far more of the factors that induce resistance in gonococci to complement-mediated killing by fresh human serum than do plasma or serum. As was previously found with serum, most of the resistance-inducing activity of guinea pig RBC lysates was found in ultrafiltrates with molecular weights of less than 5000. In contrast, and as with human serum, most of the resistance-inducing activity of human RBC lysates did not pass ultrafilters which removed molecules of less than 5000 daltons, although some active material of low molecular weight was present.
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- Physiology And Growth
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Spheroplasts of Synechococcus PCC 6301
More LessOf a number of osmotic stabilizers tested, 0·5 m-l-proline was the most suitable for the formation of spheroplasts of Synechococcus PCC 6301 with minimal loss of viability. However, even this stabilizer inhibited colony formation.
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Growth Characteristics of the Yeast Phase of Paracoccidioides brasiliensis in a Chemically Defined Medium
More LessThe growth of four clinical strains of Paracoccidioides brasiliensis was investigated using the chemically defined medium of McVeigh and Morton. Emphasis was placed upon controlling conditions of inoculum preparation, age of inoculum used, and the homogeneity of samples used for analysis. The medium was evaluated for its ability to support growth of the yeast phase of P. brasiliensis at 37 °C. Cultures were followed for 240 h and growth patterns were determined by measuring optical density, dry weight, nucleic acids and protein. The medium is excellent for growing the yeast phase of P. brasiliensis. The exponential phase lasted an average of 135 h and the stationary phase 72 h; a decline began after 207 h. This defined medium supports abundant growth of the yeast phase of P. brasiliensis and may thus prove useful in the preparation of yeast-phase antigens.
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Size Control in a Small-size Mutant of Saccharomyces cerevisiae
More LessThe kinetics of cell proliferation of a population of a small-size mutant of Saccharomyces cerevisiae were compared with those of a wild-type cell population, using time-lapse cinephotomicrography. The mean size of small-size mutant cells was approximately half that of wild-type cells at corresponding points in the cell cycle. The cycle times of small-size mutant cells were much more variable, especially for daughter cells, than those of wild-type cells. The difference in the variability of cycle times of the two strains was mainly due to the different degree of variability of their respective unbudded periods. Like wild-type cells, daughter cells of the small-size mutant were smaller and had a longer cycle time than parent cells. The small-size mutant retains a single size control over the cell cycle.
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Accumulation of Amino Acids by Saccharomyces cerevisiae Y185 with Phospholipids Enriched in Different Fatty-acyl Residues: a Statistical Analysis of Data
More LessSaccharomyces cerevisiae Y185, grown anaerobically in media containing ergosterol and palmitoleic, oleic or linoleic acids, synthesized phospholipids extensively enriched in the exogenously supplied fatty acid. A study was made of the effect of solute concentration on rates of accumulation of nine amino acids by organisms enriched in different fatty-acyl residues. Data were fitted using computer-aided statistical analysis to three equations to derive kinetic constants for accumulation. Analysis of data for two of the amino acids, namely l-threonine and l-histidine, showed different kinetics in organisms enriched in different fatty-acyl residues. Woolf-Hofstee plots for accumulation of l-threonine, as well as l-serine, showed abrupt changes in curvature at low concentrations with differently enriched organisms. Data for accumulation of both amino acids gave a significant fit to the model describing accumulation by one transport system without diffusion. Data for accumulation of l-histidine as well as l-aspartic acid best fitted a model describing accumulation by one transport system and diffusion. Values for K T and the diffusion constant, but not V max, differed only for accumulation of L-histidine in organisms with different fatty-acyl enrichments. A third model, describing accumulation by two separable transport systems, best fitted data for accumulation of l-glutamic acid and L-methionine. Data for accumulation of l-leucine, l-isoleucine and l-valine could not be fitted to any of the models. Woolf-Hofstee plots for accumulation of l-leucine and l-isoleucine by organisms enriched in oleyl or linoleyl residues were superimposable, although similar plots for accumulation of l-valine differed in shape.
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Derepression of Nitrogen Fixation in Desulfovibrio gigas and its Stability to Ammonia or Oxygen Stress in vivo
More LessNitrogen fixation (acetylene reduction) by Desulfovibrio gigas was displayed weakly and capriciously in lactate/sulphate media and sequential or continuous diazotrophic cultures were not successfully established. Washed cells from lactate-grown N-limited chemostat populations at 28 C showed reproducible anaerobic derepression of acetylene reduction, accompanied by limited growth, in low-N buffer, if sodium pyruvate + sodium sulphate were provided. Hydrogenase activity was not affected by the concentrations of acetylene used. Optimum concentrations of cells, pyruvate + sulphate, casein hydrolysate, Na2MoO4 and FeSO4 were established: peak activities of ~10 nmol C2H2 reduced min−1 (mg bacterial protein)−1 occurred with 10% (v/v) C2H2 after about 48 h; Ni2+, M2+ or derepression under Ar did not influence activity. NH+ 4 or air prevented derepression. An oxidant, usually sulphate, was essential. Thiosulphate was a poor substitute for sulphate; sulphite was apparently ineffective. Lactate, fumarate or H2 did not replace pyruvate as derepression substrate.
Pyruvate-derepressed populations showed reversible inactivation when exposed briefly to air. Activity was substantially inhibited by 10 or 100 μm-NH+ 4, reversibly at low NH+ 4 concentrations.
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Carbon Dioxide Inhibition of Photosynthetic Growth of Chlorella
More LessChlorella cultures were grown in a tubular loop reactor which facilitated both irradiation of the culture and gas mixing compared with a conventional stirred vessel with vortex aeration. Measurements of the inhibition of maximum specific growth rate (proportional to photosynthetic rate) in the tubular reactor showed that CO2 behaves as a typical inhibitory substrate at partial pressures (PCO2 ) up to 0·6 atm. The PCO2 for 50% reduction in maximum specific growth rate was 0·36 atm. At 0·6 atm there was a discontinuity in the inhibitory effect with a sharp increase in the inhibitory effect at higher PCO2 values. Cultures rapidly adjusted to step changes in the PCO2 up to 0·6 atm. At a PCO2 of 1 atm inhibition was complete but the inhibitory effect was readily reversed.
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The Effect of Food Preservatives on pH Homeostasis in Escherichia coli
More LessThe effects of cinnamic, propionic, benzoic and sorbic acids on the growth and intracellular pH of Escherichia coli were investigated. The data suggest that the potency of weak acids as food preservatives is related to their capacity to reduce specifically the intracellular pH. The data also suggest that although both the undissociated and dissociated forms of the acid cause the intracellular pH to fall, growth inhibition is due predominantly to the undissociated acid.
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Active Transport of Proline by Coxiella burnetii
More LessThe obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3·0 to 4·5, a pH range approximating that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent K m for proline transport from 45 μm to 15 μm, with the V max increasing from 3μ6 pmol s−1 (mg dry wt)−1 to 4·8 pmol s−1 (mg dry wt)−1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. d-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.
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Transport of α-Aminoisobutyrate into Trypanosoma brucei brucei
More LessThe uptake of α-aminoisobutyrate (AIB) by washed cell suspensions of bloodstream forms of Trypanosoma brucei brucei has been shown to be an energy-dependent process. No metabolism of AIB was detected under conditions leading to a 100-fold accumulation of AIB within the organism. Kinetic studies revealed that AIB uptake involved two components; that operating at low substrate concentrations had an apparent K m of 4·6 mm. Experiments with ionophores such as gramicidin and carbonylcyanide m-chlorophenylhydrazone were consistent with the AIB uptake system operating as a H+-symporter responding to the electrochemical gradient of H+, the major component of which was the membrane potential.
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A Miniature Flow Cell Designed for Rapid Exchange of Media Under High-power Microscope Objectives
More LessThe design, fabrication and use of a flow cell that allows rapid displacement of media viewed by short working distance, high power objectives are described. The cell has a small internal depth (about 0·04 cm), small volume (about 0·05 ml), and is chemically inert. It has been tested extensively in studies of tethered bacteria.
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Growth of Micro-organisms in Gel-stabilized Two-dimensional Diffusion Gradient Systems
More LessThe Szybalski wedge plate technique was modified to generate two-dimensional diffusion gradients for two environmental variables: pH value and NaCl concentration were selected in this study. After experimentation, pH values chosen ranged from 3·9 to 8·1 and salt concentrations from 23 to 79 gl−1. The pH gradients were slightly sigmoidal whilst the salt gradients were approximately linear. The reproducibility of the technique was assessed and found to be satisfactory in replicate growth experiments. A wide range of growth patterns was observed for a large variety of different bacterial types. The growth boundary and the pattern details are sufficiently distinct to suggest that the technique can distinguish between closely related species or strains.
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Induction by Ethylene of Macrocyst Formation in the Cellular Slime MouldDictyostelium mucoroides
More LessA hitherto unidentified volatile substance(s) is known to induce macrocyst formation in a strain (Dm 7) of Dictyostelium mucoroides and in a mutant (MF 1) derived from it. The properties of this substance suggested that it might be ethylene, and here it is shown that this is indeed the case. The addition of ethylene to MF 1 cells, in conditions otherwise favouring sorocarp formation, induced the formation of macrocysts. Conversely, the addition of mercury perchlorate, an absorbent of ethylene, inhibited macrocyst formation and induced sorocarp formation under conditions otherwise favouring macrocyst formation. Two inhibitors of ethylene synthesis, aminooxyacetic acid and aminoethoxyvinyl glycine, also inhibited macrocyst formation. Production and release of ethylene by D. mucoroides cells was confirmed by gas chromatography.
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Growing Hyphae of Achlya bisexualis Generate a Longtitudinal pH Gradient in the Surrounding Medium
More LessGrowing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8·5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.
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- Systematics
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A New Filamentous, Gliding Bacterium, Filibacter limicola gen. nov. sp. nov., from Lake Sediment
More LessA new, Gram-negative, multicellular, filamentous, gliding bacterium is described. The organism was isolated from the sediment of a eutrophic lake on a dilute peptone medium. The growth habit on solid media was characterized by spreading whorls of growth and spiral colonies. Filaments, 8 to 150 μm long, were composed of cylindrical cells 1·1 μm wide by 3 to 30 μm long. Junctions between individual cells within a filament were marked by constrictions. The organism was not pigmented, and therefore resembled members of the genus Vitreoscilla. Comparision with two Vitreoscilla strains showed important differences in cytochrome composition, DNA base composition, isoprenoid quinone content, and sensitivity to actinomycin D, which indicated that the isolate was more closely related to the Flexibacteriaceae than to Vitreoscilla spp. The organism did not resemble any previously described taxon of Flexibacteriaceae. On the basis of differences from both Vitreoscilla spp. and flexibacteria it is proposed that the organism be placed in a new genus, Filibacter, with the type species named as Filibacter limicola sp. nov., from its origin in sediment. The type strain of F. limicola is 1SS101 (NCIB 11923).
The organism was a strict aerobe capable of growth on defined mixtures of amino acids, and had a requirement for vitamins. Only amino acids served as substrates. The organism required particular combinations of amino acids for growth. No single amino acid or mixture of amino acids from a single biosynthetic family supported growth.
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Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the Bacterial ‘Brown Blotch’ Pathogen on Rice by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams
More LessThirty-five Xanthomonas campestris pv. oryzae,fourteen X. campestris pv. oryzicola strains and six ‘brown blotch’ pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotypic features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotypic tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii)‘Brown blotch’ strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and ‘Pseudomonas azotocolligans’ belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or pv. oryzicola strains and any phenotypic or protein electrophoretic property or clustering.
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- Short Communication
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Haemoprotein b-590 (Escherichia coli); Redesignation of a Bacterial ‘Cytochrome a1’
More LessA ‘soluble’ fraction from anaerobically grown Escherichia coli contains a haemoprotein with spectral properties, notably an α-band in the reduced form at 585 to 595 nm, similar to cytochrome a 1. Haem extraction of either the soluble preparation or whole cells yields haem b, but not haem a. In view of this, and the spectral similarities of the a 1-like component to well-known high-spin haem b proteins, we propose that the name ‘haemoprotein b-590’ be used to describe this substance and that consideration be given to the applicability of the name to certain other cytochrome a 1-like pigments in bacteria.
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Electron Microscopic Evidence of Antibody Entry into Neutrophils after Phagocytosis of Highly Virulent Group B Streptococci
P.H. Cleat, C. Wells and C.R. CoidAn electron microscopic study was undertaken of the entry of specific antibody into neutrophils containing surviving intracellular highly virulent group B streptococci after phagocytosis of the organisms had occurred. Electron micrographs are presented to demonstrate that specific antibody gains access to the ingested bacteria. This antibody binds to the surface of the streptococci, which subsequently permits the neutrophil to kill these organisms.
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