- Volume 129, Issue 8, 1983
Volume 129, Issue 8, 1983
- Biochemistry
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A C25,C25 Diether Core Lipid from Archaebacterial Haloalkaliphiles
More LessThe membrane lipids from an archaebacterial haloalkaliphile were shown to be based almost entirely on diethers containing C25 isopranyl chains. The ‘universal’ C20,C20 archaebacterial diether core lipid (2,3-di-O-phytanyl-sn-glycerol) made up only 9% (w/w) of the total isopranoid diether fraction. The rest of the isopranoid diether fraction comprised 85% (w/w) C20,C25 diether (2-O-sesterterpanyl-3-O-phytanyl-sn-glycerol) and 6% (w/w) of a novel C25,C25 diether (2,3-di-O-sesterterpanyl-sn-glycerol).
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Resistance of Clostridium perfringens to -Lactam Antibiotics Mediated by a Decreased Affinity of a Single Essential Penicillin-binding Protein
More LessBenzylpenicillin-resistant mutants of Clostridium perfringens have been isolated by in vitro selection. The sole mechanism of resistance was a decreased affinity of the highest molecular weight penicillin-binding protein (PBP1) for the antibiotic.
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The in situ Assay of Candida albicans Enzymes during Yeast Growth and Germ-tube Formation
More LessConditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0·2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1·9 and a s 0·5 of 0·6 mm, whereas in cell extracts, it had a Hill coefficient of 1·9 and a s 0·5 of 1·0 mm. The K m for ATP was 1·6 mm in cell extracts and 1·8 mm in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (s 0·5 of 2·3 mm, Hill coefficient of 4·0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.
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Distribution of a Corticosteroid-binding Protein in Candida and Other Fungal Genera
More LessUsing [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (K d) in the various isolates ranged between 8 and 19 nm; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)−1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.
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Involvement of Coenzyme A Esters in the Metabolism of Benzoate and Cyclohexanecarboxylate by Rhodopseudomonas palustris
More LessRhodopseudomonas palustris was grown on benzoate, cyclohexanecarboxylate or succinate under anaerobic or aerobic conditions. Studies of oxygen uptake by intact bacteria indicated that cyclohexanecarboxylate was metabolized aerobically by a β-oxidation sequence and that cultures grown anaerobically on benzoate also possessed this capacity. Bacteria grown on succinate were able to oxidize octanoate but not alicyclic acids. The enzymes necessary for the β-oxidation of cyclohexanecarboxylate appeared to be constitutive in both anaerobic and aerobic bacteria, the only exception being an acyl-CoA synthetase which used benzoate and some alicyclic acids as substrates. This acyl-CoA synthetase differed from the constitutive short-chain fatty acyl-CoA synthetase in that it was induced by anaerobic growth on benzoate or by aerobic growth on cyclohexanecarboxylate.
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Survey of Taurine Uptake and Metabolism in Staphylococcus aureus
More LessTaurine has been reported to be a component of the capsular polysaccharide of the encapsulated M strain of Staphylococcus aureus. This led to a study of the uptake and metabolism of [1,2-14C]taurine in a variety of encapsulated and unencapsulated S. aureus strains. Taurine was taken up by all strains studied. A discrepancy between uptake measured as depletion of radioactivity from growth medium and as cell-associated radioactivity suggested that taurine may be catabolized to CO2 in some strains. In most strains, cell-associated radioactivity was located mainly in cold TCA-soluble (pool metabolites) fractions. About 90% of the cell-associated radioactivity was present in the pool metabolites fraction in the M strain, and about 10% in hot TCA-soluble (nucleic acid-teichoic acid-capsular polysaccharide) fraction. Radioactivity in spent medium and the capsular polysaccharide-containing fraction appeared to be present as taurine in this strain. Radioactivity in the pool metabolites fraction of three of the strains examined did not chromatograph as taurine, indicating that taurine was converted into other cell metabolites. One strain incorporated radioactivity from taurine into cellular macromolecules, thus revealing a heterogeneity of staphylococcal taurine metabolism.
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The Effect of Membrane-bound β-Lactamase on Linoleic Acid Sensitivity in Staphylococcus aureus
More LessThe presence of a plasmid conferring resistance to penicillin (PC plasmid, e.g. pI258 blaI−) in Staphylococcus aureus NCTC 8325 increases the sensitivity of such a bacterium to the growth inhibitory effects of linoleic acid, whereas a plasmid conferring resistance to tetracycline does not affect linoleic acid sensitivity. The increased linoleic acid sensitivity of bacteria containing a PC plasmid may be related to the penicillinase protein itself since (i) strains having inducible penicillinase show increased sensitivity only after induction, (ii) strains in which penicillinase is directed from chromosomal or plasmid-borne genes show similar increased linoleic acid sensitivity and (iii) notwithstanding the above, the linoleic acid inhibitory effect is enhanced in a strain in which penicillinase activity is greatly reduced by a point mutation in the structural gene for penicillinase. The enhanced linoleic acid sensitivity seems to require the membrane-bound penicillinase since added extracellular penicillinase does not confer this sensitivity, and there appears to be a specific interaction between the membrane-bound penicillinase activity and linoleic acid.
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Purification and Characterization of Cytoplasmic Proteins Synthesized in the Developing Forespores of Bacillus subtilis during Sporulation
More LessForespores of Bacillus subtilis 60015 were isolated from sporulating cells at t 5 and were incubated with an amino acid mixture containing [14C]phenylalanine. Three species of 14C-labelled cytoplasmic proteins synthesized in the forespores were purified to homogeneity by gel filtration in the presence of detergents and by ion-exchange chromatography. The molecular weights of the purified proteins, called A, B and C, were estimated by SDS-PAGE as 24200, 11500 and 12700, respectively. Protein B contained remarkably high amounts of tyrosine (23·8%) and alanine (22·8%), and proteins A and C contained relatively high amounts of glutamate + glutamine, glycine, and alanine (10·4 to 13·3%). The synthesis of these proteins can provide markers for the control events in the forespore compartment during sporulation.
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The Separation of Proteins Based on Their Age, for the Study of Protein Degradation in Escherichia coli
More LessDensity labelling with 2H2O has been used in association with isopycnic centrifugation to isolate proteins of known age from cultures of Escherichia coli. The physical properties of protein samples of known age have been examined to detect a correlation between specific properties and susceptibility to degradation. No evidence of a correlation between size, charge, thermodynamic properties or amino acid composition, on one hand, and susceptibility to degradation, on the other hand, was observed following step-down conditions of growth. However, the SH content of proteins in E. coli does appear to be correlated with their susceptibility to degradation.
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Comparative Studies on Extracellular Penicillinases of the Same Structural Gene, penP, Expressed in Bacillus licheniformis and Bacillus subtilis
More LessExtracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exolarge penicillinase (mol. wt, 30500; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.
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Biosynthesis of Wax Esters in the Psychrophilic Bacterium Micrococcus cryophilus
More LessThe biosynthesis of wax esters by the psychrophilic bacterium Micrococcus cryophilus has been investigated using radio-gas chromatographic analysis of the constituent fatty acid and fatty alcohol moieties. Degradation studies, using a;-oxidation, and the kinetics of labelling from radioactive precursors show that wax ester fatty alcohols are derived from fatty acids. Based on different labelling patterns in wax esters and phospholipids formed from acetate or a saturated fatty acid supplied exogenously, two pathways of wax ester biosynthesis are proposed. It is suggested that wax esters can be derived in the main from either a cytoplasmic or a membrane-bound pool of fatty acyl-coenzyme A thioesters and alcohols, depending on whether the fatty acid is synthesized endogenously or is supplied exogenously.
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- Ecology
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Isolation of Mycoplasma and Ureaplasma Species from Raccoon Dogs (Nyctereutes procyonoides viverrinus)
More LessMycoplasma spp. were isolated from five wild raccoon dogs (Nyctereutes procyonoides viverrinus). On the basis of biochemical properties and serological tests, nine isolates were identified as Mycoplasma edwardii and four were similar to a possibly new Mycoplasma sp. represented by strain LM2 which is negative for both glucose fermentation and arginine hydrolysis. In addition, ureaplasmas were detected from these animals. Ureaplasmas were compared serologically with ureaplasma strains isolated from human, monkey, cattle, goat, sheep, cat, chicken and dog and cross-reacted with one of four serological groups of canine ureaplasmas.
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Selection of Attachment Mutants during the Continuous Culture of Pseudomonas fluorescens and Relationship between Attachment Ability and Surface Composition
More LessA strain of Pseudomonas fluorescens that had been isolated from a freshwater source on a plastic substratum was grown in continuous culture in minimal medium. The ‘adsubble’ process (adsorptive bubble separation process) was found to foam-fractionate wild-type cells from the fermenter during flow conditions. This selection pressure favoured the enrichment of two major classes of mutant, both having cell surface characteristics fundamentally different from the wild-type. The wild-type produced very little extracellular polysaccharide, whereas a ‘mucoid’ mutant, found predominantly in the aqueous-phase, produced an alginate exopolymer. The second class of mutant was isolated from the walls of the fermenter and, like the wild-type, produced little exopolymer. This mutant, with crenated colony morphology, showed increased attachment to solid surfaces compared to the wild-type and mucoid cells when assayed for attachment to polystyrene surfaces for 2 h. Outer-membrane protein, lipopolysaccharides and exopolysaccharides of the wild-type and both mutants were analysed. The results demonstrate the role of cell surface characteristics in the adaptability of the organism to micro-environments such as a solid/liquid or air/liquid interface or the aqueous phase.
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- Genetics And Molecular Biology
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Isolation of Polysomes from Nostoc sp. MAC and Translation of Messenger RNA in a Heterologous Cell-free System
Manju Gupta and N.G. CarrA method has been established which isolates polysomes from the lysozyme/EDTA-shocked cyanobacterium, Nostoc sp. MAC. In a typical preparation the total recovery of RNA as polysomes was 83%, in which 77% of the polysome fraction was present at sizes > 5-mers and 23% as 2–4-mers. Messenger RNA isolated from such a preparation of polysomes produced a 10-fold stimulation in the incorporation of [35S]methionine into polypeptides by a cell-free system of Escherichia coli. The in vitro-synthesized polypeptides were analysed on an SDS-polyacrylamide gradient gel together with in vivo-labelled proteins of Nostoc sp. MAC: seven polypeptides co-migrated with the in vivo-synthesized products. This is the first report of the expression of cyanobacterial messenger RNA in a heterologous cell-free system from E. coli; the efficiency of the system is discussed.
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Isolation and Properties of Strains of Micrococcus (Deinococcus) radiodurans Unable to Excise Ultraviolet Light-induced Pyrimidine Dimers from DNA: Evidence for Two Excision Pathways
More LessA mutant of Deinococcus (formerly Micrococcus) radiodurans (strain 302, mutant in mtcA) sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine in an attempt to isolate strains deficient in the ability to excise UV-induced pyrimidine dimers. Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers. The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively. When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant. On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles. Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302. The results indicate that wild-type D. radiodurans possesses two pathways for the excision of pyrimidine dimers and that mutational blocks in both must exist for the excisionless phenotype to be expressed.
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The Genetic Basis of Differences in Cation Chemoreception Sensitivity in Plasmodia of the Myxomycete Physarum polycephalum
More LessHybridization experiments were carried out between strains of the myxomycete Physarum polycephalum which differed in their sensitivities to the cations Na+, K+, Mg2+ and Ca2+ as determined by the threshold for changes in membrane potential on application of these ions. The sensitivities were explicable on the basis of control by three genes (mon, mag and cal) for monovalent cations, magnesium and calcium, respectively, with low sensitivity dominant. Departures from the expected ratios for three unlinked loci are explicable by high sensitivity for divalent cations reducing viability, but the additional possibility that high divalent cation sensitivity cannot be expressed in the presence of low monovalent cation sensitivity has not been excluded. Chemotactic thresholds corresponded with membrane potential thresholds.
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The Construction and Genetic Analysis of Polyploids and Aneuploids of the Pentose-fermenting Yeast, Pachysolen tannophilus
More LessTriploid and aneuploid strains of the xylose-fermenting yeast Pachysolen tannophilus were constructed. This species is a strongly homothallic organism in which the haploid phase normally predominates. The technique for producing polyploids involved prototrophic selection and interruption of the normal sequence of events leading from nuclear fusion to meiosis. Confirmation of triploidy was obtained by tetrad analysis. The haploid chromosome number of P. tannophilus was estimated to be between 5 and 7.
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Ethanol Production from Various Sugars by Strains of Pachysolen tannophilus Bearing Different Numbers of Chromosomes
More LessIncreasing chromosome number of Pachysolen tannophilus above the haploid level increased the yield of ethanol from d-xylose. There was a large increment on going from the haplophase to the diplophase, and the highest yields were obtained with either a triploid or a probable tetraploid, depending on the concentration of d-xylose. In addition, the rate of ethanol production from d-xylose and d-glucose increased, with the increment being larger on d-xylose. On d-galactose, the amount of ethanol produced within a given time also increased. The level of a by-product from d-xylose, xylitol, decreased on going from a haploid to higher ploidy, but there was no discernible trend for two other by-products, acetic acid and arabinitol. Increasing ploidy increased growth rate on d-galactose, but not appreciably on d-xylose. The altered properties on D-xylose of the strains tested were not due to increased levels of either d-xylose reductase or xylitol dehydrogenase, nor probably of alcohol dehydrogenase.
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Transfer of Nitrate Reductase Genes of the Cyanobacterium Nostoc muscorum into Rhizobium japonicum
More LessTransformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893. A spontaneous nitrate reductase deficient (Nar−) mutant (NR-6) of R. japonicum CB1809 was isolated with a frequency of 8·4 × 10−7. Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR. Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N. muscorum into this strain. This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N. muscorum DNA (with heat-treated DNA as control). Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R. japonicum CB1809 and N. muscorum.
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Transfer of an Indigenous Plasmid of Rhizobium loti to other Rhizobia and Agrobacterium tumefaciens
More LessRhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pIDl, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix− phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.
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Specification of the Conjugative Pili and Surface Mating Systems of Pseudomonas Plasmids
More LessConjugative pili were identified for representative Pseudomonas plasmids of incompatibility groups P-2, P-3, P-5, P-7, P-8, P-10, P-11, and P-13, pili for groups P-1 and P-9 having already been described in detail. FP5 pili (unclassified) were also found. In most cases pili could be characterized by electron microscopy as rigid or flexible. The majority of Pseudomonas plasmids transferred significantly better on a surface than in a liquid. Examples of all incompatibility groups were tested.
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5-Azacytidine Induces Heritable Biochemical and Developmental Changes in the Fungus Aspergillus niger
More LessTransient exposure of mycelia from Aspergillus niger to the cytidine analogue 5-azacytidine, at concentrations which do not affect the growth rate of the fungus on nearly minimal media, result in a dose-dependent, heritable change in the timing of the conidiation programme as well as heritable over-production of adaptive enzymes (glycosidases and phosphatases) and modification in the control properties of acid phosphatase. These heritable changes are induced by 5-azacytidine in a non-random way since the new phenotypes are exhibited not only by isolated clones but also by mixed populations of mycelia several life cycles (thousands of mitoses) after exposure to the drug.
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Identification of Entomocidal Toxins of Bacillus thuringiensis by High Performance Liquid Chromatography
More LessEntomocidal proteins produced by certain subspecies of Bacillus thuringiensis were isolated from purified crystals (parasporal bodies) by column chromatography using Sephacryl S-300. The crystals of most subspecies contained only one entomocidal protein (P-1), but some strains of B. thuringiensis which had been classified as subsp. kurstaki, subsp. thuringiensis, subsp. kenyae and subsp. tolworthi appeared to produce an additional protein (P-2) as a minor component of the crystal. The protein (P-2) was serologically similar to the mosquito factor previously discovered in the HD-1 strain of subsp. kurstaki. These entomocidal proteins (P-1 and P-2) were isolated and digested by trypsin. The peptides resulting from the trypsin digestion were mapped by high performance liquid chromatography (HPLC). HPLC patterns of the toxins, in particular P-1, were reliably reproducible and revealed differences in P-1 toxins between strains even in the same serotype. Analysis of P-2 by HPLC indicated that it is different from P-1 in protein structure.
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R-Plasmid-mediated Chromosome Mobilization in the Facultative Methylotroph Pseudomonas AM1
More LessChromosomal genes of the facultative methylotroph, Pseudomonas AM1, have been mobilized using the broad host-range plasmid, R68.45. Genes coding for C1 metabolism were transferred at a frequency of 10−5 to 10−4 per donor cell. The genes coding for resistance to cycloserine, phosphonomycin and streptomycin are linked to the methanol dehydrogenase gene, while the marker for thiamin auxotrophy is not.
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- Immunology
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Immunochemical Analysis of Antigenic Determinants of Chlamydia trachomatis by Monoclonal Antibodies
More LessMonoclonal antibodies to outer membrane of Chlamydia trachomatis lymphogranuloma venereum (LGV) strain L1 (440-L) were used for antigen characterization. Two separate type-specific antigenic determinants (epitopes) and one species-specific epitope were represented in the major outer-membrane protein (MOMP, molecular weight 40000) of homologous (440-L) and heterologous (IOL-1962, 810-B) L1 strains. One of the type-specific antibodies, the reactivity of which was not affected by oxidation or reduction of the antigenic sites, partially prevented the binding of species-specific antibody as determined by solid-phase radioimmunoassay. The binding of type- and species-specific antibody could be destroyed with proteolytic enzyme treatment. The reactivity of genus-specific monoclonal antibody originating from another fusion (L2, 434-Bu) was unaffected by proteolytic treatment but was sensitive to periodate, indicating the carbohydrate nature of the genus-specific epitope. Heating of antigens had no effect on the affinity of monoclonal antibodies studied.
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- Pathogenicity And Medical Microbiology
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Immunological Properties of Monoclonal Antibodies Specific for Meningococcal Polysaccharides: the Protective Capacity of IgM Antibodies Specific for Polysaccharide Group B
More LessTwo IgM monoclonal antibodies, MB32 and MB34 specific for meningococcal polysaccharide group B have been raised. Both were detectable by radioimmunoassay and agglutination, but only MB34 was effective in counter immunoelectrophoresis and complement fixation. MB34 was also far more potent than MB32 when tested for passive protection of mice infected with either Neisseria meningitidis group B or Escherichia coli K1. These data demonstrate that group B-specific antibodies do play a protective role in mice infected with these bacteria.
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Effectiveness of Phages in Treating Experimental Escherichia coli Diarrhoea in Calves, Piglets and Lambs
More LessA mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9: K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy.
A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.
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- Physiology And Growth
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Effects of Metal-depleted Media on the Growth and Morphology of Saccharomyces rouxii and on the Status of Periplasmic Acid Phosphatase
More LessSaccharomyces rouxii cells were maintained on yeast extract/neopeptone/glucose medium (YNG). Experimental medium was depleted of endogenous metal ions by repeated passage of YNG over a chelating ion-exchange resin. This treatment did not compromise the glucose or vitamin content but provided a basal medium to which essential salts could be added in defined concentrations. Accordingly it was possible to demonstrate growth requirements by this yeast species for K+, Mg2+, Zn2+ and Fe3+. Na+ would not replace K+. Additions of 1·0 μm-Ca2+, NH4+ and Na+, or 0·1 μm-Ni2+, Sn2+, Al3+, Co2+ or Cr3+ to a minimal, reconstituted medium were without effect. Cu2+ and Mn2+ additions were not required for growth but, like Mg2+, Zn2+ and Fe3+, they positively affected the biosynthesis of periplasmic acid phosphatase. Cells from iron-depleted medium had only 1 unit (g dry wt)−1 of native acid phosphatase activity although this could be stimulated 10-fold by addition of FeCl3 to washed cell suspensions before enzyme assay. Additions of 10–60 μm-FeCl3 to untreated YNG medium progressively increased the concentration of acid phosphatase after 3 d of culture to an upper value of 75 units (g dry wt)−1, and also increased the amount secreted into the growth medium. While unfortified YNG supported cells with only 28% of their acid phosphatase expressed (the bulk being subject to activation by Fe3+ in the assay medium) the cells from YNG fortified with 60 μm-FeCl3 displayed fully activated acid phosphatase. Cells from Mg2+-depleted medium exhibited bizarre, elongated cell shapes and those from Zn2+-deficient medium appeared somewhat angular by light microscopy. Fe3+-deficient cells had apparently normal morphology.
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Rotenone- and Cyanide-insensitive Respiration in Mitochondria from Neurospora crassa
More LessWhen Neurospora crassa was grown in the presence of chloramphenicol, the oxidation of NAD+-linked substrates by isolated mitochondria became largely insensitive to rotenone. It appears that chloramphenicol hindered the biogenesis of a functional rotenone-sensitive NADH-ubiquinone oxidoreductase. The rotenone-resistant pathway was able to donate electrons to both cyanide-sensitive and -resistant oxidases, indicating the operation of a branched system rather than of parallel respiratory chains. Growth of N. crassa in the presence of chloramphenicol also strongly enhanced the rates of oxidation of exogenous NADH and NADPH. This increased oxidation by mitochondria was largely insensitive to cyanide. The addition of exogenous AMP to isolated mitochondria did not specifically stimulate the electron flux through the alternative oxidase.
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Regulation of Enzyme Synthesis during the Growth of Hyphomicrobium X on Mixtures of Methylamine and Ethanol
More LessBatch cultures of Hyphomicrobium X mut 1 lacking methanol dehydrogenase activity were grown in media containing both methylamine and ethanol as carbon source. The pattern of growth did not show the characteristics of diauxic growth but methylamine was the preferred substrate. When the concentration of the methylamine in the medium fell to 9 mm (±0·8 mm), ethanol was utilized and the two substrates were then utilized simultaneously until the methylamine was exhausted from the medium. Growth then continued using ethanol. The levels of key enzymes and the Q O2 values for the two substrates supported this growth pattern and accounted for the measured uptake of the two substrates from the medium. When the wild-type Hyphomicrobium X was used in similar experiments, methylamine and ethanol appeared to be used simultaneously. However, the Q O2 values for methanol and ethanol were the same until ethanol dehydrogenase activity was induced. This suggested that the ethanol utilization observed before the induction of ethanol dehydrogenase was due to the activity of methanol dehydrogenase induced by the presence of methylamine. When this was taken into account the growth pattern obtained with the wild-type organism was the same as that seen with the mutant.
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Effect of Oxygen on the Synthesis, Activity and Breakdown of the Rhizobium Denitrification System
More LessThe synthesis, activity and breakdown of the denitrifying enzymes of Rhizobium japonicum, R. lupini and R. meliloti were found to be regulated by O2. Nitrogen oxide reductases were present in anaerobically grown and symbiotic R. japonicum, but in the case of organisms that had been grown aerobically the enzymes were induced only after a period of incubation under anaerobic conditions. Activity of the denitrification system that had been induced in aerobically grown cells was inhibited by O2. Denitrification by anaerobically grown cells and bacteroids was stimulated by 5% O2. Air inhibited denitrification completely. Little loss of denitrifying activity was shown by cells incubated in 5% O2, but cells incubated at ⩾10% O2 showed a rapid loss of denitrification activity.
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The Effect of Alkaline pH on Growth and Metabolic Products of a Motile, Yellow-pigmented Streptococcus sp
More LessStreptococcus BL 78/7 was isolated from alkaline potato-processing effluent, and resembled S. casseliflavus in morphology, motility and pigment. In aerobic conditions, at controlled pH, growth occurred between pH 5 and 11 with a maximum growth rate between pH 8 and 9 and a maximum yield at pH 8. The major metabolic products formed from glucose were lactate and acetate. The molar proportion of lactate to acetate decreased with increasing pH, from 4:1 at pH 5 to approximately 1:1 above pH 9. Growth occurred under nitrogen over the pH range from below 6 to 11, with a maximum growth rate and yield at pH 7 to 8. Lactate was the major metabolic product, with formate, acetate and ethanol also present in the molar ratio of 2:1:1. The molar proportion of lactate to formate decreased from 5:1 at pH 6 to 1·3:1 at pH 11.
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Axenic Growth of Dictyostelium discoideum Wild-type NC-4 Cells and its Relation to Endocytotic Ability
More LessA method for the axenic growth of Dictyostelium discoideum wild-type strain NC-4 is described. There were some differences between the nutritional requirements of strain NC-4 and an axenic strain Ax-2 derived from NC-4. Of the numerous components in growth media, the nature and concentration of the peptone was especially critical for axenic growth of NC-4 cells; 3% Bacteriological-peptone (Oxoid) giving the best growth rate and yield. NC-4 cells seemed to require more vitamin B12 than Ax-2 cells. Other vitamins like folic acid, biotin, and riboflavin also stimulated axenic growth of NC-4 cells. Pinocytosis was examined by the use of FITC-dextran and the results showed that the above peptone considerably enhanced the pinocytotic activity of both NC-4 and Ax-2 cells. Bacto-peptone (Difco) also enhanced pinocytosis to a similar extent, though it never supported axenic growth of NC-4 cells. Therefore, stimulation of pinocytosis may be necessary for nutrient uptake, but is not sufficient for axenic growth. The pinocytotic ability of cells decreased conspicuously during the course of development, particularly just before aggregation. The biological significance of this is discussed in relation to cell differentiation.
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Regulation of Glycogen Metabolism and Glycogen Phosphorylase in Physarum polycephalum
More LessGlycogen and glycogen phosphorylase content of microplasmodia from Physarum polycephalum were studied during carbohydrate-limited growth and spherulation, induced by starvation. The results indicate that glycogen metabolism in this organism responds most strongly to the availability of external glucose. Glycogen phosphorylase is a constitutive enzyme, present with the same specific molecular activity at all phases of growth and during transition to spherules, supporting the observation that the enzyme cannot be regulated covalently by phosphorylation-dephosphorylation. The enzyme is regulated only by metabolites, in a rather inefficient way: even during net synthesis of glycogen its degradation is not entirely stopped, resulting in a futile cycle. Its activity is merely slowed down to approximately 30% of its rate during glycogen breakdown after consumption of glucose. At this time, part of the glycogen, broken down, is apparently used for the synthesis of slime.
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The Effect of -Galactosides on the Protonmotive Force and Growth of Escherichia coli
More LessThe effect of three β-galactosides on the components membrane potential (Δψ) and pH gradient (ΔpH) of protonmotive force and growth of Escherichia coli has been examined. A good correlation between the reduction of the protonmotive force and growth inhibition was observed. Thus some galactosides had little effect on either the protonmotive force or growth while lactose diminished the protonmotive force and caused growth inhibition. This effect of lactose was dependent on the ionic composition of the growth media. In Medium A (77 mm- Na+, 85 mm- K+) lactose diminished Δψ but had no effect on ΔPH. Growth inhibition was transient at an external pH 6·0 but complete at pH 7·5. In medium KA (approximately 1 mm-Na+, 162 mm-K+) ΔpH was diminished and Δψ was not affected and consequently growth inhibition was complete at pH 6·0. In medium NA (163 mm-Na+, 20 mm-K+) lactose had little effect on Δψ,ΔpH or growth. These data support Skulachev’s hypothesis of buffering of the protonmotive force by K+ and Na+ gradients.
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Influence of the General Control of Amino Acid Biosynthesis on Cell Growth and Cell Viability in Saccharomyces cerevisiae
More LessThe general control of amino acid biosynthesis was shown to play an important role in the coordination between cell growth and cell division under amino acid limitations. Mutant strains defective in this regulatory system, as studied here mainly with mutant strain RH375 (ndr1-1), showed excessive and aberrant cell growth under mild limitation, and rapid loss of cell viability under severe amino acid limitation. Furthermore, wild-type (NDR1) cells were able to derepress, or at least maintain, levels of enzymes subject to the general control under amino acid limitations. The ndr1-1 mutant cells showed significantly decreased enzyme levels under these conditions. The loss of viability of ndr1-1 mutant cells was not due to inability to accumulate at ‘Start’ under amino acid limitation. In conclusion, we postulate that the aberrant behaviour of ndr-mutant cells is due to an inability to maintain adequate levels of amino acid biosynthetic enzymes throughout the mitotic cell cycle.
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Energy Requirement for Diazotrophic Growth of the Cyanobacterium Anabaena variabilis Determined from Growth Yields in the Dark
More LessThe cyanobacterium Anabaena variabilis CCAP 1403/13a (UTEX 1444, ATCC 29413) was grown diazotrophically in the dark in the presence of fructose with a doubling time of 25 h at 35 °C. This was 40% of the maximum rate observed in the light. Growth rates in the dark were similar with nitrate or ammonium as nitrogen sources or under diazotrophic conditions. Dark-grown cyanobacteria reduced acetylene in the dark at 25% of the rate observed for photoautotrophic cultures in the light. Heterotrophic growth yields for dark growth with different nitrogen sources were estimated from the extent of fructose-dependent growth in batch cultures. The ratio between cell carbon and fructose carbon was 0.38 in diazotrophically grown cells, 0.38 with nitrate and 0.52 with ammonia. Thus the energy requirement for growth on molecular nitrogen is similar to the energy requirement for growth on nitrate, but greater than that for growth on ammonia. Since the diazotrophic culture always had a dissolved O2 concentration of at least 80% of air saturation, this demonstrates that there are no detectable extra energy requirements for aerobic nitrogen fixation compared to nitrate reduction, such as would result from a need for respiratory protection of nitrogenase.
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- Short Communication
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Further Evidence for the Existence of a Membrane Potential in Trypanosoma brucei brucei
More LessThe distribution of 137Cs+, in the presence of valinomycin, has been used to measure the magnitude of the membrane potential (△ψ) in bloodstream forms of Trypanosoma brucei brucei. Values of the △ψ falling in the range − 100 mV to − 160 mV were observed and the maintenance of this △ψ was sensitive to certain ionophores and protonophores.
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Effects of Chloramphenicol on Cell Division in Synchronized Cells of Alcaligenes eutrophus
More LessChloramphenicol inhibited growth of asynchronous cells of Alcaligenes eutrophus. In synchronous cultures, different effects on cell division were observed, depending on the time of addition of chloramphenicol. The earlier the time of addition, the greater the inhibition of cell division, which indicates that protein necessary for cell division is synthesized at the beginning of the cell cycle.
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- Taxonomy
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Esterase Isoenzyme Variation in the Genus Saprolegnia, with Particular Reference to the Fish-pathogenic S. diclina-parasitica Complex
More LessEsterase isoenzyme patterns determined by slab gel electrophoresis were compared for nearly 60 Saprolegnia isolates, particularly from the S. diclina-parasitica complex. Consistent differences were found between S. diclina and S. parasitica isolates, with the latter being characterized by having between one and five very fast moving esterase bands that were generally absent from the former. A range of asexual (unidentifiable) isolates, taken from the vicinity offish hatcheries or from fish lesions, were also examined and their esterase isoenzymes shown to be similar to those of S. parasitica, although usually showing fewer bands. The use of esterase isoenzymes for screening potential fish pathogenic isolates of Saprolegnia is briefly compared with results obtained using oogonium morphology or cyst ornamentation, and the relative merits of each method are discussed. It is proposed that, on the basis of their distinctive cyst coat ornamentation (bundles of long ‘boathooks’) and esterase isoenzymes, fish lesion isolates can be distinguished from saprophytic isolates, and that analysis of isoenzymes should provide a useful method for the screening of potential pathogenic isolates.
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Numerical Taxonomy of Moderately Halophilic Gram-negative Bacteria from Hypersaline Soils
More LessA total of 132 moderately halophilic bacteria were isolated from hypersaline soils with a Cl- content between 2.36 and 12.72% (w/v) located near Alicante (S.E. Spain) and examined for 98 phenotypic characteristics including their response to cytological, physiological, biochemical and nutritional tests. They were submitted to a numerical analysis together with six reference strains using both simple matching (SSM ) and Jaccard (SJ ) coefficients, and cluster analysis was carried out by the unweighted pair group method of association (UPGMA), single linkage and complete linkage. With the SJ coefficient and UPGMA clustering, eight phenons were obtained at the 65% similarity level. From each phenon representative strains were chosen for the determination of DNA base composition and for electron microscopy. Bacteria belonging to phenons D, E, and F were assigned to the genus Alcaligenes. Phenon G included 27 strains assigned to Acinetobacter, but the high G + C composition (58.9 mol %) of a representative strain of this phenon suggests that it may represent a new taxon. Phenons A, B, and C were designated Flavobacterium and phenon H was Pseudomonas. The bacteria found in these environments are not related to those from hypersaline waters or normal soils.
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