- Volume 138, Issue 8, 1992
Volume 138, Issue 8, 1992
- Sgm Special Lecture
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- Biochemistry
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Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Paracoccus denitrificans
More LessThe purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium Paracoccus denitrificans were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)−1(deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent K m values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 mM, 5.9 μM, 0.47mM, 12.5 μM and 14 mM, respectively. The specific activity of purified GS was 1125 nkat (mg protein)−1(transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent K m values for L-glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, L-glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 mM, respectively. After the adenylylation of GS, the K m for L-glutamine and L-glutamate increased and reached the values of 8.0 and 27 mM, respectively. The effects of the changes in GS activity on the ammonia metabolism of Paracoccus denitrificans are discussed.
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Oxidation of L-thiazolidine-4-carboxylate by L-proline dehydrogenase in Escherichia coli
More LessL-Thiazolidine-4-carboxylate (T4C, γ-thioproline) is a toxic analogue of L-proline. T4C can be oxidized by Escherichia coli to form N-formylcysteine, which is hydrolysed to yield formate and cysteine. To determine if L-proline dehydrogenase (EC 1.5.99.8) catalyses T4C degradation, membrane fractions from E. coli were tested for T4C and proline oxidation activity. The specific activity for T4C oxidation in membranes from bacteria grown with 10 mM-proline was similar to the specific activity for proline oxidation and about 100 times that in membranes from bacteria grown without proline. Both oxidation activities were inactivated at 45 °C at the same rate. Membranes from a strain with a deletion of the putA gene encoding L-proline dehydrogenase or a strain with a putA::Tn5 insertion mutation had no detectable activity with either substrate. Although T4C was a simple competitive inhibitor of proline oxidation, proline inhibited T4C oxidation in a way that gave competitive but sigmoidal kinetics. At low concentrations, T4C induced proline dehydrogenase synthesis. Cysteine auxotrophs containing the putA:: Tn5 mutation could still use T4C as a cysteine source, and bacteria with this mutation consumed oxygen in the presence of T4C at half the control rate. These results indicate that T4C is a substrate and an inducer of L-proline dehydrogenase but suggest that E. coli also contains a second enzyme catalysing T4C degradation.
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Novel degradative pathway of 4-nitrobenzoate in Comamonas acidovorans NBA-10
A Comamonas acidovorans strain, designated NBA-10, was isolated on 4-nitrobenzoate as sole carbon and energy source. When grown on 4-nitrobenzoate, it was simultaneously adapted to 4-nitrosobenzoate and 4-hydroxylaminobenzoate but not to 4-hydroxybenzoate or 4-aminobenzoate. In cell extracts with NADPH present, 4-nitrobenzoate was degraded to 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate. Partial purification of the 4-nitrobenzoate reductase revealed that 4-nitrobenzoate is degraded via 4-nitrosobenzoate to 4-hydroxylaminobenzoate. The substrate specificity of the enzyme was narrow and NADPH was 15 times more effective as a cofactor than NADH. The results provide evidence for a novel pathway for aerobic degradation of 4-nitrobenzoate, since neither 4-hydroxybenzoate nor 4-aminobenzoate were involved in the degradative pathway.
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Investigation of an endoglucanase essential for the action of the cellulase system of Trichoderma reesei on crystalline cellulose
More LessAn investigation into the induction of the cellulase complex of Trichoderma reesei has shown that crystalline cotton cellulose was effectively degraded by the system induced by scoured cotton but not by the system induced by Solka Floc. A variety of techniques including FPLC, chromatofocusing, SDS-PAGE and isoelectric focusing (IEF) were used to separate and characterize the individual cellobiohydrolases, endoglucanases, cellobiases and glucosidases involved in cellulose degradation. A statistical comparison of the enzymic activities of the differently induced systems after resolution by anion exchange chromatography revealed much lower carboxymethylcellulase activity in one of the pooled fractions from a Solka-Floc-induced preparation, suggesting that one of the endoglucanases is either absent or present at a reduced level. IEF separations indicated that the Solka-Floc-induced system lacked an endoglucanase with an alkaline pI.
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Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)
More LessCytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.18 g ml−1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23–1.24 g ml−1) contained 60% of the lipopolysaccharide-associated, β-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.
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- Genetics And Molecular Biology
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An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002
More LessThe iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively. The genes form a dicistronic operon that is transcriptionally activated under iron-stress conditions to produce an abundant monocistronic message containing isiA and a much less abundant dicistronic message that also contains isiB. The arrangement of these genes, their transcriptional control and the relative abundance of the monocistronic and dicistronic messages produced under iron stress parallels the pattern shown by the freshwater cyanobacterium Synechococcus sp. PCC 7942. The genes for the corresponding proteins found under iron-replete conditions, CP-43 and ferredoxin, have also been cloned and sequenced. Northern blot analysis indicates that both of these genes are constitutively expressed under both iron-stress and iron-replete conditions.
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Molecular cloning, characterization and nucleotide sequence of the gene for secreted α-amylase from Xanthomonas campestris pv. campestris
α-Amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant λ library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported α-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the α-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the α-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The α-amylase synthesized in E. coli was located in the periplasm.
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Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa
More LessA 1.95 kb DNA fragment containing the aly gene from Klebsiella pneumoniae, which encodes an alginate lyase, has been ligated into the broad-host-range vector pLAFR3. Transfer of the resultant recombinant plasmid, pALY8, into mucoid and non-mucoid strains of Pseudomonas aeruginosa resulted in expression of the alginate lyase. The heterologously expressed alginate lyase, which had the same isoelectric point and substrate specificity as the native enzyme, altered the morphology of mucoid strains. Analysis of the extracellular material from mucoid strains revealed that lyase expression reduced the M r and overall yield of alginate produced. The mature form of the recombinant enzyme was the same as that produced extracellularly by Klebsiella pneumoniae; however, most of the alginate lyase was retained intracellularly by P. aeruginosa.
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p43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis
More LessThe novel mycobacterial insertion sequence IS900 was analysed by coupled transcription-translation, of both strands independently, in a cell-free E. coli extract using an exogenous promoter. This revealed only one protein product, p43, as predicted from the nucleotide sequence. The protein was readily translated in recombinant E. coli, using the tac promoter, though it did not appear as a major product by SDS-PAGE analysis. A synthetic peptide was used to generate and affinity-purify a specific anti-p43 antibody, which clearly identified the protein in recombinant E. coli. p43 was relatively stable in exponential phase and stationery phase bacteria, though a 28 kDa processed form was seen to accumulate over a period of hours. Both forms appeared in the soluble fraction of the bacterial lysate. The anti-p43 antibody also identified p43, as a 28 kDa processed product, in Western blots of protein extracts from Mycobacterium paratuberculosis, indicating a level of expression which would be unusually high for a classical transposase. These data have important implications for the relationship between IS900 and its host.
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Cloning and DNA sequence analysis of a bacteriocin gene of Serratia marcescens
More LessSerratia marcescens N28b synthesized and secreted a bacteriocin, with a molecular mass of 45 kDa, which was capable of inhibiting the growth of Escherichia coli. The expression of this bacteriocin was negligible unless induced with mitomycin C. The genes encoding the bacteriocin were cloned in plasmid pBR328. E. coli harbouring recombinant plasmid pBA189 or pBA289 expressed the Serratia marcescens N28b bacteriocin. The nucleotide sequence of the bss gene (Serratia marcescens N28b bacteriocin structural gene) was determined. The predicted amino acid sequence of the carboxy-terminal part of the bacteriocin 28b had a high degree of similarity to the pore-forming domains of colicins A, E1, B, N, Ia and Ib.
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- Pathogenicity And Medical Microbiology
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Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea-pig and human alveolar macrophages
The major extracellular enzyme of Legionella pneumophila, a metalloprotease, has been proposed as a pathogenic factor in Legionnaires' disease due to its cytotoxic, tissue-destructive, and phagocyte-inhibitory properties. The relevance of these activities depends on the production of the protease during infection, i.e. by L. pneumophila multiplying intracellularly. In this study, L. pneumophila was demonstrated to produce protease in guinea-pig and human alveolar macrophages infected in vitro. After 24 h infection, approximately 0.1 to 0.2 μg of protease per 106bacteria was measured by ELISA in culture supernatants and lysates of the infected cells, whereas no protease could be detected immediately after infection. Immunogold labelling using anti-protease antibody showed the enzyme to be located within phagosomes and distributed throughout the macrophages. Recent observations have shown that this protease could modify host defence mechanisms through inhibition of bacterial killing by neutrophils and monocytes. The intracellular production of the enzyme in infected macrophages demonstrated here further supports a role for the protease in the pathogenesis of Legionnaires' disease.
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Adhesive properties conferred by the plasminogen activator of Yersinia pestis
More LessA genomic library of Yersinia pestis EV76c created in a cosmid vector was screened for clones capable of binding type IV collagen. An unexpectedly high number of such clones was observed. One recombinant plasmid was selected for further study, and the locus controlling collagen binding was mapped by subcloning, transposon mutagenesis and exonuclease digestion. The outer-membrane protein profiles of transposon insertion mutants were correlated with phenotype to implicate a 36 kDa polypeptide in type IV collagen binding. Fine substructure restriction mapping and limited DNA sequence analysis showed the cloned locus to be identical to the locus (pla) for the plasminogen activator, previously characterized genetically and biochemically. The pla locus is resident on a 9.5 kb plasmid in wild-type Y. pestis strains. Fning of this plasmid resulted in negligible reduction in collagen-binding capacity, implying the existence of a chromosomally located determinant for collagen binding. The affinity of the plasminogen activator for collagen was relatively weak. When the cloned pla locus was introduced into E. coli, it conferred upon the cell the ability to bind to cells from a number of cell lines. Binding to glycolipids separated by thin-layer chromatography demonstrated that the receptor was a member of the globo-series of glycolipids. Since it has been reported that mutation of pla dramatically reduces virulence, we propose that this hitherto undescribed function of the gene product could contribute to the biological activities necessary for full virulence.
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Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells
More LessTwenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.
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Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica
More LessThe prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli. The gene was isolated by DNA:DNA hybridization experiments using a radioactively-labelled fragment of the homologous prn gene from Bordetella parapertussis. DNA sequence analysis reveals that the gene is capable of encoding a protein with a molecular mass of 93996 Da (P.94); this prefnsor molecule is processed to form the P.68 antigen on the surface of B. bronchiseptica. Heterologous expression of the full-length gene encoding P.94 in Escherichia coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane. Comparison of P.94 with the P.93 and P.95 prefnsors, encoding homologous proteins from Bordetella pertussis and B. parapertussis, shows a high degree (>90%) of homology. The major differences between all three proteins ocfn in the number of repeats of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.
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Heterotypic protection of mice against chlamydial salpingitis and colonization of the lower genital tract with a human serovar F isolate of Chlamydia trachomatis by prior immunization with recombinant serovar L1 major outer-membrane protein
More LessIntrauterine infection of mice with a human genital tract isolate of Chlamydia trachomatis (serovar F) resulted in salpingitis. In some cases, oviduct damage was sufficient to cause infertility due to lumenal blockage. Parenteral immunization with a purified, heterologous, recombinant major outer-membrane (rMOMP) preparation reduced the proportion of animals developing severe salpingitis by 77% compared with mock-immunized controls, but failed to reduce chlamydial colonization of the lower genital tract. In contrast, mice immunized with rMOMP directly into the Peyer's patches to stimulate mucosal immunity shed fewer chlamydiae from the vagina than controls, but showed little reduction in oviduct damage. No consistent correlation was observed between antibody levels to rMOMP in immunized mice and reduced lower genital tract colonization. Immunization with rMOMP via the presacral space, a route previously shown to stimulate mucosal immunity in the genital tract, produced high levels of circulating anti-rMOMP IgG but only traces of anti-rMOMP IgA in vaginal secretions. There was no difference in the severity of salpingitis in these animals compared with mock-immunized controls. Immunization with rMOMP conferred no protection against infertility resulting from direct inoculation of chlamydiae into the oviducts.
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The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA
More LessMycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5′-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a — 35 box, a — 10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of prefnsor rRNA secondary structure. Sequences upstream from the 5′-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for prefnsor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.
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- Physiology And Growth
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Calcium involvement in dimorphism of Ophiostoma ulmi, the Dutch elm disease fungus, and characterization of calcium uptake by yeast cells and germ tubes
More LessExogenous Ca2+, at concentrations up to 5 mM, induced partial germ tube formation in Ophiostoma (=Ceratocystis) ulmi in media normally supporting growth in the yeast-like phase. The calmodulin inhibitors calmidazolium (R24571) and trifluoperazine (TFP), and the Ca2+ionophore, A23187, suppressed germ tube formation in germ-tube-inducing medium without affecting yeast-like growth. R24571 was the most effective inhibitor, giving almost complete suppression at 3 μM. Addition of excess Ca2+ (up to 5 mM) did not reverse the inhibitory action of R24571 and only ∼ 10% of yeast-like cells formed germ tubes on addition of Ca2+ in the presence of 20 μM-TFP or 15 μM-A23187. Intracellular cAMP increased on incubation with R24571 and A23187, possibly as a result of inhibition of the cAMP phosphodiesterase. The exogenous supply of the calcium-binding agents methylhydroxybenzoate (MHB) and EGTA also suppressed germ tube formation under inducing conditions. These results confirm an involvement of Ca2+ in the yeast−mycelium transition of O. ulmi. Yeast-like cells and germ tubes of O. ulmi exhibited metabolism-dependent Ca2+ uptake which was reduced in the absence of glucose, or by the presence of KCN, the ATPase inhibitors N,N′-dicyclohexylcarbodiimide (DCCD) and diethylstilboestrol (DES), and the protonophoric uncoupler DNP, indicating dependence on the electrochemical proton gradient across the plasma membrane generated by the H+-ATPase. Germ tubes exhibited greater sensitivity to inhibitors of Ca2+ uptake than yeast-like cells, while Ca2+ uptake was competitively inhibited by Mg2+, Mn2+ and Zn2+. R24571 and A23187 inhibited Ca2+ uptake by germ tubes although TFP stimulated uptake in comparison to control cells. Ca2+ uptake by both cell types conformed to Michaelis−Menten kinetics at concentrations below ∼ 200 μM but deviated strongly above this concentration. Kinetic analysis of Ca2+ uptake by yeast-like cells and germ tubes, at Ca2+ concentrations below 100 μM, revealed that both cell types possessed Ca2+ transport systems of similar specificity, with K m values ranging between ∼ 15 and 25 μM, although germ tubes always exhibited greater Ca2+ uptake than yeast cells under similar experimental conditions, possibly a consequence of increased vacuolar compartmentation.
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A mutant of Neurospora crassa that has a long lag phase in low-calcium medium
More LessA mutant of Neurospora crassa that has a long lag phase when grown in medium that contains less than 10 μM-CaCl2 was isolated from UV-irradiated conidia. However, the rate of uptake of calcium ions by mycelium and the dependence on magnesium ions of the growth of this mutant were the same as those of the wild-type strain. The phase of the circadian clock, within the entire circadian cycle of the mutant, could not be shifted by exposure to fluorescent white light during growth in liquid medium. However, a brief incubation at high temperature resulted in a large shift in phase, and the phase response fnve for the mutant was similar to that for the wild-type strain. The mutant strain had the same sensitivity to light as the wild-type strain with respect to carotenoid synthesis. The relevant mutation may affect the process involved in transduction of signals from the light-perception system to the clock mechanism and this process may be related to calcium homeostasis in Neurospora cells.
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Glucose-induced activation of the plasma membrane H+-ATPase in Fusarium oxysporum
More LessAddition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. lini triggered activation of the plasma membrane H+-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in vivo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent K m of about 3.2 mM for ATP whereas the activated enzyme showed an apparent K m of 0.26 mM. In addition, the pH optimum of the H+-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced H+-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. lini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5.0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H+-ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H+-ATPase. These results suggest a possible causal relationship between the activity of F. oxysporum var. lini plasma membrane H+-ATPase and the intracellular level of cAMP.
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Coupling between the respiratory chain and the luminescent system of Vibrio harveyi
More LessEffects of monovalent cations on luminescence and respiratory activity were studied in the marine luminous bacterium Vibrio harveyi. Maximum oxygen uptake was observed in the presence of Na+over the pH range tested (6.5–8.5). At alkaline pH, effects of monovalent cation on luminescence were similar to those on the oxygen uptake. Although KCN addition caused a marked increase in luminescence, the enhanced luminescence with Na+was still greater than that with Li+. However, at acidic pH, K+increases luminescence more than Na+does. These results indicate that there is not only a competitive but also a cooperative relationship between luminescence and respiration. The respiratory NADH oxidase in the membrane fraction of V. harveyi showed some distinctive characters which are unique to the respiratory-dependent primary Na+pump, suggesting the possibility of coupling between the Na+pump and the luciferase system. This was also supported by the results from CCCP-resistant growth and luminescence at alkaline pH. The coupling mechanisms between luminescence and respiration in V. harveyi are discussed.
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Glycogen and poly-β-hydroxybutyrate synthesis in Spirulina maxima
More LessThe effect of different growth conditions on the glycogen and poly-β-hydroxybutyrate (PHB) content of the cyanobacterium Spirulina maxima is described. Under photoautotrophic growth conditions without any nutrient limitation, S. maxima exhibited a glycogen content of between 7.1 and 10.7% of cell dry wt, whereas PHB was undetectable. When S. maxima was grown under mixotrophic conditions in the presence of acetate, the intracellular PHB concentration increased to more than 3% of dry wt, while glycogen content remained within the range of 5 to 6% of cell dry wt. Nitrogen starvation favoured glycogen accumulation (up to 60 to 70% of dry wt), while the PHB content remained low (up to 0.7% of dry wt), even after prolonged nitrogen starvation. Inhibition of protein synthesis, induced by addition of azaserine, led to the accumulation of glycogen (up to 52% of cell dry wt) but did not stimulate PHB synthesis. Under phosphorus-limited growth conditions, glycogen and PHB accumulated (up to 23% and 1.2% of cell dry wt, respectively) only after the exhaustion of intracellular phosphorus reserves. Shifting the culture from low to high light irradiance induced a rapid accumulation of glycogen (up to 34% of cell dry wt after 9 h) but did not induce PHB synthesis. Results are discussed in terms of the metabolic significance of PHB synthesis in cyanobacteria, and suggest that this polymer acts exclusively as a disposal mechanism to eliminate excess reducing equivalents.
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The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria
More LessThe aim of this investigation was to perform an extensive screening using HPLC and 13C-NMR spectroscopy to disclose the spectrum of osmolytes produced by aerobic heterotrophic and anoxygenic phototrophic eubacteria. The most predominant solutes detected within a wide range of marine and halophilic micro-organisms were two recently discovered tetrahydropyrimidines ectoine and hydroxyectoine, which were synthesized in response to osmotic stress.
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Production of cellulolytic and xylanolytic enzymes during growth of the anaerobic fungus Piromyces sp. on different substrates
More LessPiromyces sp. strain E2, an anaerobic fungus isolated from an Indian elephant (hindgut fermenter) was tested for its ability to ferment a range of substrates. The fungus was able to use bagasse, cellobiose, cellulose, fructose, glucose, lactose, mannose, starch, wheat bran, wheat straw, xylan and xylose. Formate and acetate were the main fermentation products after growth on these substrates. The amount of carbon found in the fermentation products of cultures, in which substrate digestion was complete averaged 88.5 mM, or 59% of the carbon offered as substrate. No growth was observed on other substrates tested. Lactose, starch, cellobiose and filter paper cellulose were good inducers of cellulolytic and xylanolytic enzymes. Cellulolytic and xylanolytic enzymes were produced constitutively by Piromyces strain E2, although enzyme activities were generally lower after growth on glucose and other soluble sugars. Complex substrates (bagasse, wheat bran, and wheat straw) were good inducers for xylanolytic enzymes but not for cellulolytic enzymes. The extracellular protein banding pattern after SDS-PAGE was therefore only slightly affected by the growth substrate. Identical β-glucosidase and endoglucanase activity patterns were found after growth on different substrates. This indicated that differences in enzyme activities were not the result of secretion of different sets of isoenzymes although it remains possible that the relative amount of each isoenzym produced is influenced by the growth substrate.
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The possible role of ADP-ribosylation in sporulation and streptomycin production by Streptomyces griseus
More LessMutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces griseus IFO 13189, a streptomycin-producing strain. One (strain no. 4), which had significantly reduced ADP-ribosyltransferase activity, was analysed in detail. Mutant 4 displayed a conditional phenotype with respect to cultivation temperature. At 30 °C, it exhibited severely reduced ability to produce aerial mycelium (on solid medium) and submerged spores and streptomycin (in liquid culture), but this ability was fully restored at 25 °C. The mutant produced A-factor normally, regardless of cultivation temperature, and exhibited normal ability to accumulate ppGpp intracellularly. SDS-PAGE analyses of cellular proteins labelled by [32P]NAD revealed that an ADP-ribosylated protein with a molecular size of 44 kDa, which appeared in sporulating cultures of the parent strain, was missing from the mutant grown at the non-permissive temperature (30 °C). Genetic analysis showed that the aba mutation conferring resistance to 3-aminobenzamide was tightly linked to the altered phenotype. Failure to ADP-ribosylate certain cellular protein(s), presumably due to the aba mutation, may be responsible for impaired differentiation in this mutant.
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Peptidoglycan biosynthesis in Escherichia coli: variations in the metabolism of alanine and D-alanyl-D-alanine
More LessThe in vivo functioning of the alanine/D-alanyl-D-alanine pathway of Escherichia coli was investigated by determining prefnsor pool levels and specific enzyme activities under various growth conditions. Cells grown on D- or L-alanine showed several remarkable features compared with cells grown on other carbon sources: 10-fold higher values of the D-alanyl-D-alanine and the UDP-MurNAc-pentapeptide pools, a 240-fold increase of the alanine racemase activity, and the absence of bacteriolysis after treatment with D-cycloserine at high concentrations (50 μg ml−1). In cells grown on glucose, D-cycloserine (1 μg ml−1) led to depletion of the D-alanyl-D-alanine pool and to lysis, which was efficiently antagonized by chloramphenicol. A threefold increase of the dipeptide pool was observed when cells were treated with chloramphenicol alone. The alanine racemase activity was lowest in glucose-grown cells and the D-alanine: D-alanine ligase and D-alanyl-D-alanine-adding activities were the same whatever the carbon source. Molecular masses of 53–56 kDa and 56–60 kDa were estimated for the partially purified inducible alanine racemase and D-alanine: D-alanine ligase respectively.
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Diversity in surface features of Cytophaga johnsonae motility mutants
More LessTwenty-eight non-spreading mutants of the gliding bacterium Cytophaga johnsonae were generated by different approaches and subsequently studied to determine whether they possessed traits commonly assumed to be associated with such mutants. Many non-spreading variants of C. johnsonae purportedly possess a static cell surface as opposed to the dynamic one of the motile parent strain. A moving cell surface has been supposed to be responsible for an array of traits associated with motile cells, but missing in non-motile cells. Phage sensitivity, chitin digestion and the ability of cells to move latex beads over their surfaces are some of the alleged motility-dependent traits. Also, it has been reported that non-spreading mutants possess a cell surface that is less hydrophobic than that of the parent strain. We characterized our collection of mutants in relation to the above mentioned traits to determine whether these characteristics indeed required a moving cell surface. Our findings showed that neither phage sensitivity nor chitin digestion were motility-dependent. In addition we noted that non-spreading mutants could possess surfaces more (rather than less) hydrophobic than the motile parent strain.
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Volumes and issues
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Volume 170 (2024)
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Volume 132 (1986)
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Volume 131 (1985)
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Volume 130 (1984)
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Volume 129 (1983)
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Volume 128 (1982)
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Volume 127 (1981)
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Volume 126 (1981)
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Volume 125 (1981)
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Volume 124 (1981)
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Volume 123 (1981)
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Volume 122 (1981)
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Volume 121 (1980)
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Volume 120 (1980)
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Volume 119 (1980)
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Volume 118 (1980)
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Volume 117 (1980)
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Volume 116 (1980)
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Volume 115 (1979)
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Volume 114 (1979)
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Volume 113 (1979)
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Volume 112 (1979)
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Volume 111 (1979)
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Volume 110 (1979)
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Volume 109 (1978)
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Volume 108 (1978)
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Volume 107 (1978)
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Volume 106 (1978)
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Volume 105 (1978)
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Volume 104 (1978)
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Volume 103 (1977)
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Volume 102 (1977)
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Volume 101 (1977)
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Volume 100 (1977)
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Volume 99 (1977)
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Volume 98 (1977)
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Volume 97 (1976)
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Volume 96 (1976)
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Volume 95 (1976)
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Volume 94 (1976)
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Volume 93 (1976)
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Volume 92 (1976)
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Volume 91 (1975)
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Volume 90 (1975)
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Volume 89 (1975)
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Volume 88 (1975)
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Volume 87 (1975)
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)