- Volume 129, Issue 10, 1983
Volume 129, Issue 10, 1983
- Biochemistry
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The Absence of Quinoprotein Alcohol Dehydrogenase in Acinetobacter calcoaceticus
More LessIt is shown that the unusual NAD(P)+-independent quinoprotein alcohol dehydrogenase, said previously to be responsible for oxidation of ethanol during growth of Acinetobacter calcoaceticus LMD 79.39, was in fact isolated from an unidentified organism which contained cytochrome c and which has now been lost. Several genuine strains of A. calcoaceticus do not contain cytochrome c nor do they contain a quinoprotein alcohol dehydrogenase. The enzyme responsible for ethanol oxidation in these bacteria is an inducible NAD+-linked alcohol dehydrogenase.
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Studies on Some Enzymes of Alginic Acid Biosynthesis in Azotobacter vinelandii Grown in Continuous Culture
More LessWhen a mutant of Azotobacter vinelandii was grown in continuous culture the amount of exocellular polysaccharide produced was dependent on both the dissolved oxygen tension (d.o.t.) and the carbon source: sucrose supported alginate synthesis in phosphate-limited medium whereas sorbitol did not. Changes in the specific activities of two of the key enzymes of alginate biosynthesis (phosphomannose isomerase and GDPmannose pyrophosphorylase), measured in extracts of cells grown with sucrose under a range of d.o.t. values, were reflected by the observed changes in alginate production; the activity of GDPmannose dehydrogenase was unchanged. A similar correlation between the specific activities of these enzymes and the rate of alginate production was observed during a transition from sorbitol to sucrose as the sole carbon ource, but in this experiment the activity of GDPmannose dehydrogenase also increased with increasing alginate production. After prolonged continuous cultivation on sucrose the mutant gradually lost the ability to produce alginate. The key enzymes of alginate biosynthesis could not be detected in extracts of this non-alginate-producing strain, which had also lost the ability to encyst. These results support the suggestions that alginate formation is controlled by derepression of key biosynthetic enzymes and that alginate plays an important role in encystment.
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Inhibitors of Synthesis of Lipid-linked Saccharides also Inhibit β-Glucan Synthesis by Cell-free Extracts of the Fungus Saprolegnia monoica
More LessGlucan synthetases from cell-free extracts of Saprolegnia monoica synthesize (1 → 3)-β or (1 → 4)-β-glucans according to the assay conditions. Bacitracin and flavomycin, inhibitors of the synthesis of lipid-linked saccharides, were efficient inhibitors of glucan synthesis [90% inhibition for (1 → 3)-β-glucans, 60% inhibition for (1 → 4)-β-glucans]. These antibiotics also inhibit glycosyl transfer to different lipids. The glucan synthesis pathway does not seem to involve glycolipid intermediates and the inhibition produced by the antibiotics is probably due to their effect on enzymes rather than to depletion of lipid acceptors.
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Multiple Forms of Polygalacturonase in Apple and Carrot Tissue Infected by Isolates of Botrytis cinerea
More LessIsoelectric focusing of extracts of carrot root and apple fruit parenchyma infected by each of three isolates of Botrytis cinerea showed the presence of forms of endopolygalacturonase (PG) with isoelectric points (pI) of about 4·5 (a minor peak) and 8·3–8·8. One isolate gave the hitherto unreported peak of pI 8·8 in both tissues, whereas the other two isolates gave a peak of 8·3. The molecular weights of the PGs of pI 8·3–8·8 were estimated to be about 30000. The variation in pi probably reflects the variability of B. cinerea. No evidence of specific enzymic adaptation to host tissues was shown by the ability of PGs to cause changes in permeability of carrot and apple parenchyma.
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The Intracellular Localization of Pseudomonas aeruginosa Lectins
More LessThe localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5′-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10–13%) and a considerable activity (38–4%) of 5′-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human eiythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.
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Role of O-Acetyl Groups in the Lipopolysaccharide Receptor for Rhizobium Phage 1P
More LessRhizobium trifolii lipopolysaccharide (LPS) preparations lost their receptor properties for phage 1P after deacetylation by alkali. LPS treated with acetylesterase isolated from Lactobacillus plantarum showed a simultaneous decrease in the amount of acetyl groups and phage receptor activity. The content of O-acetyl groups in the LPS of a phage-resistant mutant was significantly decreased when compared to that in LPS from the parent phage-sensitive strain 24SM. Phage 1P could cleave acetyl groups from the extracted LPS without any detectable formation of reducing groups. Binding of phage 1P to the LPS receptor was irreversible. It was demonstrated by electron microscopy that phage DNA was ejected in vitro in the presence of LPS from R. trifolii 24SM but not of that from the phage-resistant mutant.
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Sub-cloning of the Wild-type proAB Region of the Escherichia coli Genome
More LessThe genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23·3 kilobases of genomic DNA. proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA. The pro operon is contiguous with the gene phoE. Hybridization experiments showed no homology between proAB of E. coli and the other regions of the E. coli genome or with the DNA of several other bacterial species.
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Partial Purification and Characterization of Two Enzymes Involved in Isovaleric Acid Synthesis in Clostridium bifermentans
More LessConversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing α-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 ± 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8·6: the apparent K m for leucine was 7·0 mm. Activity was totally inhibited by 1 mm-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated α-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography en DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the K m for α-ketoisocaproic acid was 4·2 mM and activity was optimal around pH 8·0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group. FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mm.
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Purification and Properties of Extracellular Glucosyltransferases from Streptococcus mutans Serotype a
More LessExtracellular glucosyltransferases (sucrose: 1,6-α-d-glucan 3-α-and 6-α-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6·20 and 5·86 i.u. mg−1, respectively, exhibited slightly different isoelectric points (pI 4·5 and 4·2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5·5 and the same K m values of 1·3 mm for sucrose and of 83 μm-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-α-d-glucans with 20 and 24·5 mol% 1,3,6-branch points, respectively. Both are therefore Afunctional enzymes.
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Stimulation of Phosphofructokinase from Phycomyces blakesleeanus and Some Other Fungi by Micromolar Concentrations of Fructose 2,6-bisphosphate
More LessThe phosphofructokinase in crude extracts of Phycomyces blakesleeanus required the presence of ammonium salts, AMP or fructose 2,6-bisphosphate for its activity. The enzyme had slightly sigmoidal kinetics with respect to fructose 6-phosphate as substrate. It was slightly inhibited by high ATP concentrations and by citrate. Fructose 2,6-bisphosphate stimulated the Phycomyces blakesleeanus phosphofructokinase; the K m for fructose 6-phosphate decreased, the inhibition by ATP was completely relieved and the affinity for the activator ammonia was increased. AMP also stimulated the catalytic activity but there was poor co-operation with fructose 2,6-bisphosphate. Preliminary experiments showed that fructose 2,6-bisphosphate also stimulated the phosphofructokinase from Mucor rouxii, Neurospora tetrasperma and Agaricus bisporus.
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- Development And Structure
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Isolation and Characterization of the Outer Membrane from Vibrio parahaemolyticus
More LessThe outer membrane of Vibrio parahaemolyticus strain 3283-61 (serotype O2:K3) was isolated from blebs released upon spheroplast formation, in the presence of lysozyme and EDTA, by isopycnic sucrose density gradient centrifugation. SDS-PAGE of the outer membrane fraction prepared from cells grown in nutrient broth containing 3% (w/v) NaCl revealed five major proteins, designated a to e, with apparent approximate molecular weights: a, 44000; b, 36 000; c, 33500; d, 26500; e, 22000. An increase in NaCl concentration in the growth medium resulted in an increase of proteins b and c, whereas a decrease to 0·5% (w/v) induced two additional major proteins with respective molecular weights of about 35000 and 32000. Proteins a and b appeared to be loosely associated with the peptidoglycan layer since they were largely retained after extraction with 2% (w/v) SDS at 50 C for 30 min. Proteins c and/or e may play a role in phage VP1-receptors since phage-resistant mutants derived from strain 3283-61 had significantly diminished amounts of both proteins. The major outer membrane proteins varied in number and molecular weight in strains of V. parahaemolyticus belonging to different K-serotypes.
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- Genetics And Molecular Biology
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Contribution of the Symbiotic Plasmid to the Competitiveness of Rhizobium leguminosarum
More LessFour different symbiotic plasmids from Rhizobium leguminosarum were introduced into three different recipient strains that lacked plasmid-linked symbiotic determinants. The twelve synthetic strains so constructed were each tested for competitiveness against a standard reference strain. The recipient strain and the introduced symbiotic plasmid contributed about equally to competitiveness in forming root nodules on pea plants: there was also significant interaction between strain and plasmid, although this was much less important than the main effects. Competitiveness for growth on the legume root surface (the rhizosphere) was attributable entirely to the recipient strain; the introduced plasmid had no significant effect.
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Water Relations of Erwinia chrysanthemi: Intracellular and Extracellular Pectate Lyase Production
More LessWhen Erwinia chrysanthemi was grown in a sodium polypectate/yeast extract/salts medium adjusted with sorbitol to water activity (aw ) values of 0·990 and 0·980, extracellular pectate lyase (PL) production was repressed, whereas intracellular PL levels were not affected. Inorganic solutes (NaCl. LiCl, KCl, Na2SO4 and a NaCl/KCl/Na2SO4 mixture) at 0·990 aw strongly stimulated the intracellular levels of PL (6-to 17-fold greater than the control), whereas extracellular levels were only slightly increased. Intra-and extracellular PL levels were not affected by LiCl, NaCl and KCl at 0·995 a w. A NaCl/sorbitol mixture (0·990 a w) completely inhibited extracellular but not intracellular PL production. Lowering of the aw of cultures during mid-exponential growth phase with NaCl (0·990 a w) resulted within 30 min in a 70-fold increase in intracellular PL activity. When sorbitol was used in similar experiments, extracellular PL production was inhibited to a greater extent than intracellular levels.
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The Genetic Location of Three Mutations Impairing Penicillin Production in Aspergillus nidulans
More LessThree mutations impairing penicillin production in Aspergillus nidulans, npeB, npeC and npeD, have been located on linkage groups III, IV and II, respectively, and positioned relative to other loci on these chromosomes.
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Heterokaryosis in Fusarium tricinctum and F. sporotrichioides
More LessHeterokaryons were formed in intra- and interspecific crosses between Fusarium sporotrichioides and F. tricinctum auxotrophs. Segregant homokaryons were evaluated for trichothecene toxin production in culture. Results were consistent with nuclear control of toxin synthesis. The sexual compatibility of auxotrophs and 30 additional F. tricinctum sensu Snyder & Hansen strains was tested. Perithecial production was restricted to crosses between Florida isolates pathogenic to English ivy (Hedera helix). The linkage of several auxotrophic markers was determined by analysis of progeny of certain crosses. No T-2 toxin was produced by sexually compatible F. tricinctum isolates.
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Heterokaryon Incompatibility and Interspecific Hybridization between Verticillium albo-atrum and Verticillium dahliae Following Protoplast Fusion and Microinjection
More LessInterspecific and intraspecific heterokaryons of Verticillium albo-atrum and Verticillium dahliae were formed by protoplast fusion or microinjection. Diploid formation was increased 1000-fold following either treatment. Combinations of auxotrophic strains which normally never form heterokaryons were obtained with both techniques. The results suggest that a variety of incompatibility factors exist in Verticillium. Compatibility and incompatibility in their strict sense did not apply to the strains used in this work, and the terms ‘heterokaryon-former’ and ‘heterokaryon non-former’ have been introduced instead. The compatibility functions are assumed to be nuclear and somehow associated with cell wall formation or components affecting the cell wall. Temperature had a profound effect on both the morphology and the nutritional marker ratio of interspecific heterokaryons. Although the frequencies of diploid formation for intc specific and intraspecific diploids were very similar, the corresponding haploidization frequencies for these diploids were markedly different. An examination of random aneuploid conidia showed that spontaneous haploidization and mitotic crossing-over occurs in interspecific diploids of Verticillium. The genetic segregation of interspecific diploids grown in the presence of haploidizing agents suggested that the two fungi are closely related and that substantial chromosomal homology may exist between them.
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Acquisition and Maintenance of Enterotoxin Plasmids in Wild-type Strains of Escherichia coli
More LessEnterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.
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Evidence for Shorter Average O-Polysaccharide Chainlength in the Lipopolysaccharide of a Bacteriophage Felix 01-sensitive Variant of
More LessProlonged culturing in the laboratory has resulted in the formation of a stable derivative of the smooth Group E bacterial strain, Salmonella anatum A1, that is sensitive to both the R-core-specific bacteriophage Felix 01 and O-polysaccharide-specific bacteriophage ε15. The variant strain, designated S. anatum A1-1, exhibits a normal number of irreversible binding sites for ε15 but the relative quality and/or accessibility of those sites appears to be diminished. Infectious ε15 phage particles are released more rapidly from S. anatum A1-1 than from its parent under acidic pH conditions known to interfere with the phage DNA ejection step.
The purified lipopolysaccharide (LPS) of S. anatum A1-1 exhibits a reduced rhamnose/hep-tose ratio in chemical assays. Fractionation of this LPS on SDS-urea-polyacrylamide gels followed by silver staining reveals a narrower range of O-polysaccharide chain lengths relative to that of the parent (0 to, 20 vs. 0 to 40 repeating units, respectively).
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Use of the Escherichia coli Transposon Tn1000 (γδ) to Generate Mutations in Bacillus subtilis DNA
More LessPlasmid pHM2 contains a Bacillus subtilis spoIIA gene and is able to replicate in both Escherichia coli and B. subtilis. The plasmid was mobilized at low frequency by the E. coli F plasmid. Nearly 30 % of the mobilized plasmids contained an insert of Tn1000 (γδ). Fourteen of the inserts were in the B. subtilis DNA portion of pHM2. Of these, two adjacent inserts abolished expression of the plasmid spoIIA gene in B. subtilis. From the map positions of flanking inserts that do not abolish spoIIA gene expression, it is estimated that the gene is probably not more than 700 bp long.
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The Pattern of Protein Synthesis in spoIVC Mutants of Bacillus subtilis Resuspended in Sporulation Medium
More LessTwo spoIVC mutants of Bacillus subtilis were labelled with [35S]methionine either at the time of resuspension in sporulation medium or 1, 2 or 3 h later, and radioactive proteins were detected after cell extracts had been subjected to two-dimensional gel electrophoresis. The mutants completed almost all of the changes in protein synthesis that occur in the wild-type in these conditions. A heavily labelled protein was found in the mutants that has also been observed in a spoO mutant but does not occur in the wild-type.
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- Pathogenicity And Medical Microbiology
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Physicochemical Surface Properties and Phagocytosis by Polymorphonuclear Leucocytes of Different Serogroups of Salmonella
More LessSalmonella isolates belonging to different serogroups have been analysed with respect to physicochemical surface properties and interaction with human polymorphonuclear granulocytes (PMNs). Most (22/34) recent isolates of the different serotypes showed hydrophilic surface properties and little if any negative charge accompanied by resistance to phagocytosis by PMN similar to the old laboratory S strains Salmonella typhimurium 395MS and Salmonella minnesota S99 (main group). However, all isolates belonging to the serogroups Cl (5 isolates), E4 (2), O43 (1), and one out of three E1 isolates (C1/E4 group) differed from the main group. In aqueous biphasic partition in dextran-polyethyleneglycol (PEG) systems the bacteria in the main group accumulated in the PEG-rich phase to 55–97%, those in the C1/E4 group to less than 10%, and R-mutants only to 1–2%.
The bacteria in the C1/E4 group displayed a negative surface charge and a susceptibility to phagocytosis by PMNs that were greater than those for strains in the main group but much lower than those shown by the R-mutants. Bacteria belonging to serogroup C1 also displayed a significant susceptibility to hydrophobic interaction. The results are discussed in relation to the pathogenicity of salmonella.
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Vero Cell Toxins in Escherichia coli and Related Bacteria: Transfer by Phage and Conjugation and Toxic Action in Laboratory Animals, Chickens and Pigs
More LessSixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT−. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli. strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.
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Fructosyltransferase Activity of a Glucan-binding Protein from Streptococcus mutans
More LessStreptococcus mutans serotype c produces several extracellular proteins which bind to affinity columns of immobilized glucans. The proteins are three distinct glucosyltransferases and another glucan-binding protein (molecular weight 74000) which is now shown to be a fructosyltransferase. This enzyme is antigenically distinct and genetically independent of two other fructosyltransferases produced by the same organism. A mutant is described which lacks the glucan binding fructosyltransferase and has defective ability to form adherent colonies in the presence of sucrose. Although the production of glucans from sucrose results in the glucan binding protein becoming bound to the bacterial surface, and hence perhaps contributing to adherence, the fructans synthesized by the enzyme do not appear to contribute to this phenomenon.
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Protoplast and Cytoplasmic Membrane Preparations from Streptococcus sanguis and Streptococcus mutans
More LessProtoplasts were prepared from Streptococcus sanguis and some S. mutans serotypes by use of lysozyme (EC 3.2.1.17) under particular conditions: cells had to be grown in dl-threonine (20 mm) and harvested in early exponential phase. The efficiency of protoplast formation was enhanced by two additional steps: plasmolysis (in 12% PEG), prior to addition of lysozyme, and a swirling phase, after the enzymic action. This procedure allowed us to obtain clean protoplasts, with only 0·5% contamination by bacterial cell walls. Up to 90% protoplast lysis was obtained in 0·5 m-NaCl. Cytoplasmic membrane purification was achieved by centrifugation on a glycerol cushion.
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- Physiology And Growth
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Evidence for the Existence of Two Homoserine Dehydrogenases in Serratia marcescens
More LessStrain D-315 was isolated from a wild-type strain of Serratia marcescens as a methionine-sensitive mutant. In this mutant, methionine-mediated growth inhibition was reversed by threonine or homoserine. The homoserine dehydrogenase activity of strain D-315 was about 20% lower than that of the wild-type and was not inhibited by threonine. The methionine-sensitive mutation was located in the thr region on the chromosome, indicating that strain D-315 lacked homoserine dehydrogenase I, whose activity is inhibited by threonine and whose synthesis is multivalently repressed by threonine plus isoleucine. In a methionine bradytroph derived from strain D-315, homoserine dehydrogenase activity was high during growth in the absence of methionine and low during growth in the presence of excess methionine. Strain D-413, a homoserine auxotroph derived from strain D-315, had no detectable homoserine dehydrogenase activity, indicating that S. marcescens had homoserine dehydrogenase II, whose synthesis is controlled by methionine-mediated repression. The gene coding for homoserine dehydrogenase II was located in the metB-argE region. Strain TA-191, having homoserine dehydrogenase I and lacking the other isoenzyme, was constructed by transduction. This strain was sensitive to threonine-mediated growth inhibition.
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Effects of Culture Density on the Kinetics of Germ Tube Formation in Candida albicans
More LessThe relationship between culture density or phase of growth at 24.5 °C and the ability of Candida albicans to form germ tubes when shifted to 37°C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.
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Low Redox Potential Promotes Sulphide- and Light-dependent Hydrogen Evolution in Oscillatoria limnetica
More LessAnoxygenic photosynthetic electron transport from sulphide, culminating in either H2 evolution or CO2 photoassimilation, was shown to include the segment from plastoquinone to ferredoxin in the cyanobacterium Oscillatoria limnetica. Both sulphide-dependent H2 evolution and CO2 photoassimilation were inhibited by plastoquinone analogues. In the former reaction, the block was bypassed by reduced N,N,N′,N′ -tetramethyl-p-phenylenediamine (TMPD). The link between this segment of electron transport and the hydrogenase enzyme was shown to limit the rate of sulphide-dependent H2 evolution. The rate of flow of electrons through this pathway was lower than would be expected either from the amounts of available enzyme, as measured by the in vitro oxidation of reduced methyl viologen, or from rates of electron transport to CO2 photoassimilation. When the strong reductant sodium dithionite was added to intact cells, the resulting low redox potential significantly improved photosynthetic sulphide-dependent H2 evolution. Hydrogenase activity in cell free extracts was similarly affected by dithionite. It is suggested that ambient redox potential controls electron flow through the hydrogenase, so that surplus reducing power is removed via H2 evolution.
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Buffer and Salt Damage to the Filamentous Cyanobacterium Anabaena cylindrica
More LessThe commonly used buffer HEPES was found to cause severe disintegration of the cyanobacterium Anabaena cylindrica, as measured by filament breakage, cell disruption, phycocyanin release and nitrogenase inhibition. The effect became increasingly severe as the buffer concentration was increased above 10 mm. The observed cell damage does not appear to be unique to HEPES, similar observations being made with Tris/HCl, sodium phosphate, sodium sulphate and sodium chloride. It appears that the cells are very sensitive to ionic strength.
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Synthesis of Adenylate Nucleotides by Mollicutes (Mycoplasmas)
More LessCultures of the Mollicutes (mycoplasmas) Acholeplasma laidlawii B, Acholeplasma morum, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma fermentans and Mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in pH and glucose concentration, and concentrations of ATP, ADP, AMP, lactate and pyruvate. The adenylate energy charge (ECA), the mean generation time, and the number of nmol of ATP (mg dry weight)−1 were calculated for cultures in the mid-exponential growth phase. The maximum cell concentrations ranged from 0·2 1010 to 5·0 1010 c.f.u. ml−1. Doubling times ranged from 0·34 to 3·29 h. The fermentative, non-arginine-requiring A. laidlawii B, A. morum, and M. gallisepticum, as well as the fermentative, arginine-requiring M. fermentans, utilized glucose and produced lactate and pyruvate. The non-fermentative, non-arginine-requiring M. bovis neither utilized glucose nor produced lactate or pyruvate. The non-fermentative, arginine-requiring M. arginini utilized glucose, but did not produce lactate or pyruvate. At mid-exponential growth phase, the average ECA of A. laidlawii B was 0·90, a value similar to that reported for Spiroplasma citri and other bacteria. In contrast, the average ECA of A. morum and the four Mycoplasma species was 0·70. In A. laidlawii B at mid-exponential growth phase, ATP accounted for 97% of the total adenylate nucleotide pool. At the same stage of growth, the average cellular ATP concentration of the other Mollicutes was significantly lower, ranging from 45 to 63% (P < 0·01). Excluding A. laidlawii B, the Mollicutes were relatively energy deficient during their mid-exponential growth phase. The diminished metabolic capacity may be related to the association of Mollicutes with living cells and perhaps to the cytopathic effects of these micro-organisms.
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‘Methylobacterium ethanolicum’: a Syntrophic Association of Two Methylotrophic Bacteria
More LessTwo methylotrophic bacteria having similar cell morphologies have been isolated from cultures of ‘Methylobacterium ethanolicum’ grown on methane. One of these, strain POC, is an obligate methanotroph containing the serine pathway and Type II intracytoplasmic membranes, which appears to be a ‘Methylocystis’ species. The second organism, strain H4-14, can fix N2 and grows on a variety of substrates, including methanol, formate, ethanol, succinate, fructose and H2 + CO2; during growth on methanol, carbon is assimilated via the Calvin-Benson cycle. Strain H4-14 appears to be a Xanthobacter species. Mixtures of the two organisms formed stable associations during growth on methane, consisting of approximately 5–20% H4-14 and 80–95% POC. Strain POC excreted biotin, which was required by strain H4-14. Our results suggest that ‘Methylobacterium ethanolicum’ cultures grown on methane consist of a syntrophic association of two methylotrophs.
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Regulation of the Metabolism of Some Alkylated Amines in the Yeasts Candida utilis and Hansenula polymorpha
K. B. Zwart and W. HarderThe initial metabolism of some methylated and ethylated amines, which were used as a nitrogen source but not as the sole carbon source by the yeasts Candida utilis and Hansenula polymorpha, involved a peroxisomal amine oxidase which produced ammonium ions, hydrogen peroxide, and formaldehyde or acetaldehyde. The aldehydes so formed were either oxidized via their corresponding carboxylic acids or, depending on the organism and the aldehyde, also partly assimilated into cell material. The synthesis of amine oxidase, which was paralleled by the development of peroxisomes in the cells, was repressed in the presence of ammonium ions and derepressed under nitrogen limitation. Amines were not required as inducers of enzyme synthesis. Utilization of ethylated amines, but not of methylated amines, as a nitrogen source resulted in a significant increase in cell yield. In both yeasts ammonium ions were assimilated mainly by way of NADPH-dependent glutamate dehydrogenase. The activity of this enzyme increased drastically in cells grown under ammonium or amine limitation or, under carbon limitation, in the presence of amines as the sole source of nitrogen. Under the latter conditions free ammonium was not detectable in the culture supernatant, while the amount of amines utilized was just sufficient to account for the amount of cell material produced. This indicated that during growth with amines as the nitrogen source the physiological condition that the cells experience is in fact one of ammonium limitation. Our results suggested that the rate of amine oxidation was determined by the intracellular concentration of ammonium and determined via repression of amine oxidase synthesis. Due to this control and the high nitrogen to carbon ratio of amines, sustained growth of the methylotrophic yeast H. polymorpha on methykmine and of both species on ethylamine as a carbon source is not possible, even though these organisms are able to grow on the related compounds methanol and ethanol.
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Purification and Characterization of Flagella from the Alkalophile Bacillus firmus RAB
More LessFlagella from Bacillus firmus RAB, an alkalophilic bacterium, were purified to homogeneity. The flagella were shown to consist of a single protein subunit (flagellin) with an apparent molecular weight of 40000. The amino acid composition of B. firmus RAB flagellin was similar to that of other bacilli except that the former had far fewer basic amino acids. The paucity of basic amino acics may render the flagella more stable at external pH values as high as 11·0.
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- Short Communication
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Cyclic AMP-dependent in vitro Activation of Trehalase from Dormant Phycomyces blakesleeanus Spores
More LessThe activity of trehalase in a crude extract of dormant Phycomyces blakesleeanus spores increased 10- to 20-fold during incubation in the presence of cyclic AMP and ATP. A phosphorylation of the enzyme was probably involved, as a more pronounced activation was obtained when cyclic AMP was replaced by the catalytic subunit of beef heart protein kinase. The γ-thio derivative of ATP could replace ATP in the activation but was quantitatively less effective. The effective concentrations of cyclic AMP and ATP were comparable to their likely intracellular concentrations; thus phosphorylation could be the in vivo mechanism of trehalase activation during the activation of dormant spores by heat or acetate.
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- Taxonomy
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Intra- and Intergeneric Similarities of the rRNA Cistrons of Alteromonas, Marinomonas (gen. nov.) and Some Other Gram-negative Bacteria
More Less14C-labelled rRNA was prepared from Alteromonas macleodii ATCC 27126 and from Alteromonas haloplanktis ATCC 14393. 3H-labelled rRNA was isolated from Alteromonas vaga ATCC 27119 and Alteromonas putrefaciens ATCC 8071 colony type tl. These rRNAs were hybridized under stringent conditions with filter-fixed DNA from various Alteromonas strains and from organisms of marine origin and/or with mol % G + C values in the range 40 to 50. Each hybrid was described by its Tm(e) and percentage of rRNA binding. From rRNA similarity maps and Tm(e) dendrograms the following conclusions were drawn. The genus Alteromonas is very heterogeneous and consists of four rRNA branches: (1) Alt. macleodii alone; (2) the Alt. haloplanktis cluster, containing most of the named Alteromonas species and a number of organisms which should be renamed Alteromonas (‘Pseudomonas marinoglutinosa’, ‘Pseudomonas nigrifaciens’, ‘Pseudomonas atlantica’ ATCC 19262, ‘Pseudomonas carrageenovora’, ‘Pseudomonas piscicida’, and several unnamed alginolytic bacteria); we propose to limit the genus Alteromonas to the former two clusters; (3) Alt. putrefaciens, the rRNA cistrons of which resemble those of the Vibrionaceae and are as different from the above two Alteromonas rRNA branches as are those of the Vibrionaceae, the Enterobacteriaceae and Aeromonas; ‘Pseudomonas rubescens’ belongs to this branch and Alteromonas hanedai seems to be a remote relative; (4) Alteromonas communis and Alt. vaga constitute another, separate rRNA branch; in conjunction with their special phenotypic features, we propose to create a new genus, Marinomonas, for them. The exact taxonomic position of ‘Alteromonas thalassomethanolica’ could not be established.
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Bullera crocea and Bullera armeniaca, Two New Yeasts from Fruit and Vegetables
More LessTwo new ballistosporogenous yeasts, Bullera crocea and Bullera armeniaca, are described. Bullera crocea was isolated from strawberries, cauliflowers and watercress, and B. armeniaca from cauliflowers and cabbage. Unlike B. alba, B. dendrophila, B. piricola and B. tsugae, these new species could not utilize lactose; B. crocea was distinguishable from B. armeniaca by the ability of the former to utilize melibiose.
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Comparison of Ureaplasmas from Sheep and Goats with Ureaplasma diversum and U. urealyticum
More LessUreaplasmas isolated from sheep and goats were compared by immunofluorescence with antisera prepared in calves and by PAGE of polypeptides labelled by growth in the presence of [35S]methionine. The ovine and caprine strains constituted two groups defined by serology and polypeptide composition that were not related to the animal species from which they originated. Strains representing these two groups were compared with Ureaplasma urealyticum (human isolates) and U. diversum (bovine isolates). They could not be classified with either but were more similar to the U. diversum strains.
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Comparative Study of Streptococcus mutans Extracellular Glycosyltransferases by Isoelectric Focusing
More LessExtracellular glycosyltransferases from 17 strains of Streptococcus mutans were examined by analytical isoelectric focusing. Three kinds of glucosyltransferase: highly-branched-1,6-α-d-glucan synthetase, 1,3-α-d-glucan synthetase and 1,6-α-d-glucan synthetase, were excreted from serotype a, d and g strains. The enzymes of serotype a strains were distinguishable from those of serotypes d and g by differences in their pI values. Serotype c, e and f strains excreted basic glucosyltransferase and acidic fructosyltransferase. Serotype b strains also excreted the glucosyl-and fructosyltransferases, but the pI values were different from those of the enzymes from the other serotypes. Thus, S. mutans strains could be divided into four groups by analytical isoelectric focusing of glycosyltransferases which corresponded well to the four genetic groups.
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