- Volume 139, Issue 12, 1993
Volume 139, Issue 12, 1993
- Review Article
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- Biochemistry
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Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species
More LessDifferent mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp. and Hyphomicrobium sp. Sulphate liberation from MMS in Agrobacterium sp. M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp. the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase. In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode. MMS-grown Agrobacterium sp. M3C and Hyphomicrobium sp. MS223 oxidized MMS with consumption of 0·5 mol O2 per mol of substrate, but they were unable to oxidize methanol. By repeatedly challenging MMS-grown Hyphomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO2− 4-liberation stopped although excess MMS was available. SO2− 4-release resumed immediately when O2 was re-admitted to the electrode chamber. Thus liberation of SO2− 4-from MMS in the oxygen electrode was dependent on the continuing availability of O2. Hyphomicrobium sp. MS223 therefore closely resembled Agrobacterium sp. M3C in its obligatory requirement for O2 in MMS degradation. Unlike Agrobacterium sp. M3C, Hyphomicrobium sp. MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0·5 mol O2 per mol of substrate) but not MMS. Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp. MS223 but did not affect oxidation of MMS by MMS-grown cells. Thus Hyphomicrobium sp. MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS. These results are consistent with the absence of methanol from the pathway for biodegradation of MMS by Hyphomicrobium sp. MS223 and suggest that in at least some Hyphomicrobium spp. an oxidative mechanism initiatesbiodegradation.
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Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium Rhodobacter capsulatus ElFl
More LessGlutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium Rhodobacter capsulatus ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome c and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes’ radii of 6·18 and 7·32 nm could be separated by gel-filtration of protein solutions made in 0·5 m-KCl, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes radius of 6·80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by l-glutamate and NADP+.
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Incomplete citric acid cycle obliges aminolevulinic acid synthesis via the C5 pathway in a methylotroph
More LessThe enzymic activities of the citric acid cycle and the connected pathway of 5-aminolevulinic acid (ALA) formation in the methylotroph Methylophilus methylotrophus (strain AS1) have been studied. The organism has the enzymes required for conversion of pyruvate to 2-oxoglutarate. Of these, isocitrate dehydrogenase is unusual because of its preference of NAD as coenzyme over NADP. In addition, the segment of the cycle that oxidizes 2-oxoglutarate to oxaloacetate is incomplete, lacking 2-oxoglutarate and succinate and malate dehydrogenase activities. Furthermore, alternative routes of 2-oxoglutarate oxidation to succinate are undetectable. The enzymes of the glyoxylate cycle are also absent. This suggests that the cycle in M. methylotrophus has no catabolic role and is purely biosynthetic. We also show that M. methylotrophus uses the C5 pathway of ALA formation. Cell-free extracts can convert glutamate to ALA in an ATP-, NADPH- and tRNA-dependent manner via the intermediate formation of Glu-tRNAGlu and glutamate 1-semialdehyde. Consistent with the absence of a detectable route by which it could synthesize succinate, M. methylotrophus cannot generate ALA from succinyl-CoA and glycine, the pathway found in mammalian cells and yeast.
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Wall formation by Candida albicans yeast cells: synthesis, secretion and incorporation of two types of mannoproteins
More LessThe mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with β-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.
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Glucose utilization and lactate production by Helicobacter pylori
More LessThe transport and incorporation of d-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize d-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3·24 mmol min−1 (g protein)−1, and the rate of incorporation into the cellular mass was 1·06 μmol h−1 (g protein)−1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.
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Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Penicillium notatum
D. Pitt and J. C. BarnesCytosolic free calcium concentration [Ca2+]c of protoplasts from Penicillium notatum was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5·8–7·0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5·0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7·2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca2+ was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 μmol to 50 μmol (kg cell water)−1, during attainment of the steady state after 30 min, at which time [Ca2+]c was unchanged at 20 nm but increased to 182 nm at 2–6 h exposure to 2·5 mm-Ca2+. Broadly similar changes in [Ca2+]c were found in protoplasts derived from mycelium samples exposed to Ca2+ over the same period of time. The location of Ca2+ was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca2+ for 6 h, Ca2+ was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2–5 μm-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca2+. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4–6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca2+-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca2+-induction of the sporulation process.
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Characterization of the 1,3-β-glucan synthase of Aspergillus fumigatus
More Less1,3-β-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors - GTP, NaF, sucrose and EDTA - added during the extraction procedure, were essential for optimal 1,3-β-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a K i of 1·42 mm and 0·3 mm for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (K m for UDP-glucose = 1·9 mm). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.
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Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins
More LessSUMMARY: The CWLA lytic amidase of Bacillus subtilis 168 was purified and antisera raised against the purified protein. No expression of cwlA could be demonstrated under any conditions by the use of the antisera and cwlA::lacZ fusion analysis. Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA. Both of these inducible lysins were found to be encoded by prophage PBSX. Prophage SPβ was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA. The smaller of the two PBSX enzymes was purified and found to be an N-acetylmuramyl-l-alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera. The potential origin of cwlA and its possible relationship to the other phage lytic enzymes are discussed.
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- Development And Structure
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Influence of Zn2+ on yeast-mycelium dimorphism and exopolysaccharide production by the fungus Aureobasidium pullulans grown in a defined medium in continuous culture
More LessThe yeast-mycelium dimorphism of Aureobasidium pullulans was studied in continuous culture in a defined medium. At a constant dilution rate (0·08 h−1) the morphological status of the culture could be controlled by the input concentration of Zn2+. As the input concentration of Zn2+ was increased (in intervals from 0 to 7·6 μm) the culture shifted from a zinc-limited to a carbon-limited state. In this interval the culture gradually passed through three growth regimes based on morphology and concentration of exopolysaccharide and biomass. The first growth regime was found when the input concentration of Zn2+ was kept below 0·45 μm. Growth in this regime was zinc-limited and more than 90% of the biomass was in the yeast growth form. An increase in the input concentration of Zn2+ in this growth regime led to a proportional increase in both the biomass and the concentration of exopolysaccharide. When the input concentration of Zn2+ was varied between 0·45 μm and 0·80 μm a second growth regime could be detected where simultaneous limitations in two nutrients were recognized. Although the carbon source (glucose) was exhausted an increase in the input concentration of Zn2+ led to a proportional increase in the steady-state biomass concentration. The increase in biomass concentration was at the expense of exopolysaccharide production, which gradually decreased. The culture, still being primarily limited by Zn2+, remained in the yeast growth form. In a third growth regime (input concentration of Zn2+ above 0·80 μm) no increase in the steady-state biomass was seen when the input concentration of Zn2+ was increased. The concentration of residual glucose and exopolysaccharide was close to zero, and no further carbon could be diverted to an increase in the biomass. Glucose was the primary limiting nutrient. Increasing the input concentration of Zn2+ in this growth regime led to a gradual increase in the mycelial growth form at the expense of the yeast growth form. More than 90% of the biomass was in the mycelial growth form when an input concentration of Zn2+ of 7·6 μm was used.
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- Genetics And Molecular Biology
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The cloning and characterization of the arom gene of Pneumocystis carinii
The arom gene, encoding a single polypeptide that catalyses five consecutive steps of the pre-chorismate aromatic amino acid biosynthetic pathway, has been cloned from the opportunistic pathogen Pneumocystis carinii. There is a single open reading frame of 4788 bp which includes an intron of 45 bp that does not introduce a stop codon into the sequence. Thus, the derived amino acid sequence consists of 1581 residues, which is highly homologous to all fungal AROM proteins studied to date. These data support the view that P. carinii is a fungus and imply that its aromatic amino acid biosynthesis is conventionally organized.
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Identification of structures containing polyphosphate in Helicobacter pylori
More LessSUMMARY: For the first time polyphosphate (poly P) granules have been detected in Helicobacter pylori organisms colonizing the gastric antrum as well as in organisms isolated from the same tissue. Poly P granules showed typical sublimation characteristics during exposure to the electron beam and chipped out of ultrathin sectioning. A prominent phosphorus signal was identified using elemental specific electron microscopy such as electron energy loss spectroscopy (EELS) and was localized to at least three different locations: the cytoplasm, the flagellar pole and in association with the cell membrane. Intracytoplasmatic structures had a diameter of 0·05–0·2 μm, whereas the structures near the flagellar pole were much smaller (0·02 μm). The membrane-associated phosphate aggregates were visible only after staining with Pb(NO3)2 or with electron spectroscopic imaging (ESI). Poly P granules seem to be important energy and phosphorus stores and it is thought that they participate in the regulation of various and distinct metabolic processes of H. pylori.
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Pisatin resistance in Dictyostelium discoideum and Neurospora crassa: comparison of mutant phenotypes
More LessThe pea phytoalexin pisatin, at its inhibitory concentration, was shown to have two distinct inhibitory effects on amoebae of the cellular slime mould Dictyostelium discoideum. One effect was cytolytic and was demonstrable even in non-growing cells whereas the second effect was observed only under conditions favourable to growth. Pretreatment with a sublethal concentration of pisatin induced the amoebae to acquire resistance to both these effects. Mutations in nysC that alter membrane sterols and confer resistance to the polyene antibiotics nystatin and pimaricin blocked resistance to the growth-associated inhibitory effect but did not affect acquisition of resistance to the cytolytic effect. The nysB sunD double mutant HK412 displayed a partially constitutive resistance to the cytolytic effect but, like the nysC mutants, was blocked in the acquisition of resistance to the growth-associated inhibitory effect. Pisatin-treated cells incubated in pisatin-free medium lost their ability to grow on pisatin-containing medium much more rapidly than they lost resistance to the cytolytic effect of pisatin. These results suggest that the induction of pisatin resistance may involve the turning-on of independent resistance mechanisms against the two inhibitory effects of pisatin. This could account for our inability to isolate pisatin-resistant mutants in a single step. The Neurospora crassa erg1 and erg3 mutants that have altered membrane sterols and are nystatin resistant displayed sensitivity to pisatin. The pisatin-sensitivity phenotype of the erg mutants was used in selections to identify complementing plasmids from an ordered Neurospora genomic library. The association of pisatin sensitivity with membrane sterol alterations in both D. discoideum and N. crassa supports the hypothesis that mechanisms underlying nondegradative pisatin resistance are evolutionarily conserved.
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Developmental and cAMP-mediated regulation of glycogen phosphorylase 1 in Dictyostelium discoideum
The Dictyostelium discoideum glycogen phosphorylase-1 (gp-1) exhibits a complex pattern of developmental expression in which differential temporal regulation of enzyme activity, protein levels and mRNA levels is observed. This pattern of expression implies that gp-1 regulation occurs at multiple levels, probably involving both transcriptional and post-transcriptional events. Post-translational control of gp-1 activity, in effect, actually regulates the protein from a developmental perspective. In this report we have examined several facets of this regulation. We show that addition of exogenous cAMP to cells in suspension culture caused changes in gp-1 enzyme activity and mRNA levels that are identical to those observed during normal development, suggesting that cAMP is involved in the regulation of gp-1. Exogenous cAMP could regulate gp-1 mRNA expression at concentrations as low as 1·0 μm. cAMP regulation of gp-1 mRNA appeared to occur through a mechanism that required intracellular cAMP signalling. We identified regions of the promoter necessary for gp-1 expression by using gp-1 promoter deletions to drive the expression of a luciferase reporter gene. Results of these experiments suggested that developmental and cAMP-mediated changes in gp-1 mRNA levels were the result of alterations in transcription. The promoter analysis also suggested that a vegetative specific element is located between −785 and −1894 nucleotides from the transcriptional start site. Elements necessary for maximal developmental and cAMP-mediated expression appear to be located between −1153 and −1894 nucleotides from the cap site. Sequence elements located between −180 and −1153 appear to be required for a basal level of late developmental expression.
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Cloning, mapping and conjugal mobility of pLPG36, a common plasmid from Legionella pneumophila serogroup-1
More LessA plasmid designated pLPG36 was isolated from the naturally occurring Legionella pneumophila serogroup-1 and purified by CsCl buoyant density centrifugation. A restriction map of this 58 kb plasmid was constructed and provided the basis for cloning four BamHI fragments into the unique BamHI site of pUC18. The four recombinant plasmids were investigated for the mobilization function in Escherichia coli strains. Only one of these, pFLJ2, was mobilized by the IncP plasmids RP4, pRK231 and R702, but not by plasmids pSa, R40a, R387, pN3 or R16. The derivative plasmid pFLJ2 was mobilized more efficiently by R702 than by RP4 or pRK231. By genetic and deletion analysis, the mobilization region of pLPG36 was located to a 6 kb EcoRI fragment of the plasmid.
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Sequencing and analysis of the divergon comprising gtaB, the structural gene of UDP-glucose pyrophosphorylase of Bacillus subtilis 168
More LessNucleotide sequencing revealed that gtaB, the structural gene of UDP-glucose pyrophosphorylase (EC 2.7.7.9), is part of a divergon-like genetic entity. The latter consists of two monocistronic operons gtaB and orfX, transcribed from a 245 bp regulatory region, each encoding an acidic protein with a molecular mass of 33·0 and 42·6 kDa, respectively. gtaB is transcribed from a distal PA promoter, and a proximal PB promoter which is negatively controlled by the Sin protein. Sin-mediated transcriptional attenuation and enhancement of PB and PD, respectively, suggest that these promoters control functions which antagonize each other. Transcription of orfX is mediated by a PA promoter. The regulatory region comprises four ATGAAA hexamers, present as two inverse repeats. Protein GtaB exhibits high homology to analogous prokaryotic enzymes, while OrfX shows 55·4% homology with the product of Escherichia coli o389, which is part of a regulatory unit involved in sugar processing. Mutations gtaB515 and gtaBg100, which define different bacteriophage adsorption patterns, were sequenced. They are transitions leading to substitution of amino acids which occupy conserved positions, and are thus likely to be part of an enzyme active site. The nature of the possible receptors for defective bacteriophages PBSY and PBSZ is discussed.
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Effects of new mutations in the spoIIAB gene of Bacillus subtilis on the regulation of σF and σG activities
More LessThe spoIIAB gene of Bacillus subtilis encodes an inhibitor of σF, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation. The SpoIIAB protein can probably also inhibit a closely related sigma factor σG, which determines the later phase of prespore-specific transcription. We have isolated two new missense mutations in the spoIIAB gene. spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of σG. In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit σF. All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors. The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of σG activity.
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Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity
More LessChlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase β subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the β subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.
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Cloning and nucleotide sequence of an extracellular α-amylase gene from Aeromonas hydrophila MCC-1
More LessA gene encoding the extracellular α-amylase of Aeromonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Escherichia coli JA221 carrying pCA101 revealed that approximately 60% of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an α-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete α-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several α-amylases were also present in A. hydrophila α-amylase at the corresponding positions.
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Cloning of an endo-(1→4)-β-glucanase gene, celA, from the rumen bacterium Clostridium sp. (‘C. longisporum’) and characterization of its product, CelA, in Escherichia coli
More LessA genomic library of Clostridium sp. (‘C. longisporum’ ATCC 49440 in the host Escherichia coli was screened for endo-β-glucanases, and plasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 3620 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1→4)-β-glucanase, CelA, assigned to family A4. N-terminal amino acid sequence determination revealed that pCM64 encoded the full-length celA gene, including a signal sequence, while pCM4 carried a 5ʹ-truncated celA gene expressed as an N-terminal fusion protein, CelAΔNʹ, without a signal sequence. CelA was secreted into the periplasm in E. coli. In this organism, proteolytic cleavage of CelA at or near a putative linker region resulted in the appearance of two active polypeptides of molecular masses 57 and 47 kDa. The former was the full-length enzyme, while the latter consisted of the catalytic domain from which the cellulose-binding domain (CBD) had been removed (CelAΔCBD). The intracellularly-located CelAΔNʹ was not subject to proteolytic degradation. The pH and temperature optima of CelA were pH 4·8 and 43 °C, respectively. CelA hydrolysed barley β-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cello-oligosaccharides (cellohexaose and cellopentaose); cellotetraose was the smallest substrate degraded completely.
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Kinetics of secretion of recombinant acid phosphatase by Myxococcus xanthus: a sensitive probe for the assay of protein translocation through the envelopes
More LessThe gene encoding a periplasmic pH 2·5 acid phosphatase (appA) from Escherichia coli was placed under the control of an inducible promoter and integrated into the chromosome of Myxococcus xanthus. The majority of the AppA protein synthesized in M. xanthus accumulated in the periplasm whereas about 30% was secreted into the medium. The kinetics of AppA secretion through the ‘outer envelopes’ (i.e. periplasm + outer membrane) were followed after AppA induction. The results suggest that AppA crosses these envelopes by a mechanism involving diffusion and show that the periplasmic accumulation of AppA and envelope permeability are decreased by mutations that decrease the secretion of native proteins in M. xanthus.
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Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis
More LessA 5·4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orfl-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 °C.
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Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies
An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20–35 times in the B. ovis genome and 5–15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53·4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on Xbal-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods. As ISs have been shown to be implicated in chromosomal rearrangement, we propose that the chromosomal polymorphism revealed by PFGE and high copy number of IS6501 observed in B. ovis may be related to the presence of an active IS in this species.
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Cloning and characterization of a tryptophanase gene from Enterobacter aerogenes SM-18
A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory element for transcription. A partial open reading frame homologous to the 5ʹ end of E. coli tnaB was observed at the 3ʹ-flanking region of tnaA. These genes may thus constitute an operon as in E. coli.
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- Immunology
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Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis
More LessBecause partial protection against reinfection is induced by experimental infection in the guinea-pig model of genital chlamydial infection, we sought to induce immunity by immunization. Female guinea-pigs were immunized subcutaneously with the major outer-membrane protein (MOMP) and the 61 kDa cysteine-rich outer-membrane protein (61 kDa) of the agent of guinea-pig inclusion conjunctivitis (GPIC) eluted from SDS-polyacrylamide gels (SDS-MOMP, SDS-61 kDa). Post-immunization sera and secretions contained antibodies to the SDS-purified proteins at high titre as measured by immunoblotting, whereas enzyme immunoassays (EIA) using whole elementary bodies as antigen showed significantly lower titres (P < 0·001). Likewise, blastogenic responses of peripheral mononuclear cells to GPIC elementary bodies were weak. Animals immunized with SDS-MOMP and SDS-61 kDa were fully susceptible to intravaginal challenge, as were control animals immunized with buffer without protein. Another group of animals were immunized with material prepared by extraction of chlamydial outer-membrane complexes with octyl β-d-glucopyranoside (OGP) and dithiothreitol, which consisted largely of MOMP (OGP-MOMP). In contrast to the SDS-MOMP group, sera and secretions in the OGP-MOMP group showed high titres in EIA, and high titre antibodies to MOMP by immunoblot; however, most animals also had antibodies to 61 kDa, 72 kDa and ca. 84 kDa outer-membrane proteins. OGP-MOMP animals were partially protected against genital challenge as evidenced by low inclusion scores compared to control animals, although duration of infection measured by culture isolation was similar to controls. Immunoblot analysis of sera from immunized animals and from a group of immune animals post-infection was performed using recombinant fusion peptides containing the four variable domains of MOMP. No consistent differences in reaction patterns were observed when sera from protected and non-protected animals were compared. Thus, a highly refined outer-membrane preparation is capable of producing partial immunity to genital infection. Further study is required to determine whether the protection is due to MOMP itself or to other outer-membrane proteins found in small amounts in the OGP-MOMP immunogen. The results suggest the possibility that discontinuous MOMP epitopes could play a role in inducing a protective immune response in the guinea-pig model, a concept that requires further evaluation.
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Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae
The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from α-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100–1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.
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- Pathogenicity And Medical Microbiology
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Molecular characterization of the coagulase-negative staphylococcal surface flora of premature neonates
More LessA single point study was conducted to determine which surface sites best represent the density and composition of the coagulase-negative staphylococcal (CNS) colonizing flora in premature neonates. Five different surface sites of six randomly selected neonates hospitalized in a neonatal intensive care unit (NICU) for a month were examined. The individual strains and their clonal organization within CNS species were identified using restriction endonuclease fingerprinting of whole chromosomal DNA and ribosomal RNA genes. Cultures of the scalp, umbilicus, foot, nose and rectum were collected and quantitatively processed. Ten colonies were typed per surface culture. The most dense CNS colonization was noted on the umbilicus (mean 1·2 × 104 c.f.u. cm−2), foot (mean 1·6 × 103 c.f.u. cm−2) and nose (mean 1·7 × 103 c.f.u. cm−2) of NICU neonates. Scalp and rectum were scarcely colonized. Of all the CNS surface isolates, S. epidermidis accounted for 77·7% (219/282) and S. haemolyticus, S. warneri and S. capitis accounted for 20·6% (58/282), 1·4% (4/282) and 0·4% (1/282), respectively. Colonization of each surface site comprised a maximum of five different strains representing four CNS species. Overall, five clones of S. epidermidis, two of S. haemolyticus, one of S. warneri and one of S. capitis were noted among the 282 isolates. The most predominant were two clones of S. epidermidis and one of S. haemolyticus; they accounted for 94% (265/282). Cultures from the foot and scalp represented the most heterogeneous CNS colonization of the five sites examined. Based on our findings of the existence of multiple strains of CNS at individual surface sites of NICU patients, we concluded that a minimum of five isolates be examined per surface culture to provide a comprehensive overview of the CNS colonizing flora.
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The aggregation of human platelets by Lactobacillus species
More LessThe ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0·25±01·and 20·4±3·2 min and the percentage aggregation ranged between 70±2·6 and 104±13·5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0·1 m-Tris/HCl (pH 8·5) at 37 °C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 μg) completely inhibited platelet aggregation by 8/9 of the homologous strains. A comparison of the platelets from four donors showed that an increase in the lag phase by 50% required 11·5, 14·8, 33·3 or 115 μg of extract, indicating a variability in donor response to aggregation by lactobacilli. The data indicate that platelet aggregation by lactobacilli may be an important contributory factor in IE. The potential to cause IE is present in the general population of oral lactobacilli and due regard should be taken when strain selection is made for probiotic purposes.
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Detection of immunoglobulin-G-binding proteins in Streptococcus suis
More LessThis study was undertaken to search for the presence of immunoglobulin G (IgG)-binding proteins in Streptococcus suis, an important swine pathogen. Whole bacterial cells were incubated with human or pig IgG conjugated to gold particles and examined by transmission electron microscopy. Cells of some S. suis strains were labelled as were cells of the positive control strain, Staphylococcus aureus Cowan I. Binding of pig and human IgG to five different bacterial species of group D streptococci, to reference strains representing the 29 capsular types of S. suis, and to 12 S. suis capsular type 2 strains was then examined using Western blotting. All strains interacted with pig and human IgG, although the binding profiles were slightly different. A 52 kDa protein was observed in all capsular types of S. suis. This protein, absent in other group D streptococcal species, was observed in all capsular type 2 isolates originating from diseased or clinically healthy pigs, and was shown to bind human IgG-Fc fragments. The IgG-binding activity was also observed in the culture supernatant and was sensitive to proteolysis.
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Biological activity of a peptidoglycan extracted from Leptospira interrogans: in vitro studies
More LessPeptidoglycan (PG) has been isolated from some species of spirochaetes, including Leptospira interrogans. Although leptospiral PG has been chemically characterized, no study has been carried out on its potential biological activity. Since PG of Treponema and Borrelia is biologically active both in vivo and in vitro, we investigated the capacity of a leptospiral PG preparation to induce relevant biological effects. PG extracted from L. interrogans strain Teramo was mitogenic at 0·1 μg ml−1 for human peripheral blood mononuclear cells (PBMC) since it increased the PBMC fraction positive for Ki-67, an antigen expressed by human proliferating cells; at 4 μg ml−1, PG was able to induce complement consumption and to stimulate leucocyte phagocytosis and the metabolic burst of resting as well as phagocytosing leucocytes. These findings indicate that Leptospira PG may play a role in modulating the immunocompetent cell functions and suggest that PG can contribute to the host response during Leptospira infection.
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Heat-inducible ATP-binding proteins of Candida albicans are recognized by sera of infected patients
More LessFour proteins from Candida albicans extracts have been isolated by ATP affinity chromatography. These proteins were found to be at elevated levels in extracts of cells raised from 25 °C to 37 °C, but were present at low levels in cells grown at 25 °C. The molecular masses of the proteins (38–42 kDa, 66–68 kDa, 70–72 kDa and 74–76 kDa) correspond to the published sizes of C. albicans heat-shock proteins. Three of the four proteins were recognized by the sera of patients with oral and/or oesophageal C. albicans infections, with the 70–72 kDa protein reacting in all cases tested. Binding of antibodies to two of the other proteins (38–42 kDa and 74–76 kDa) differed from patient to patient. IgA antibodies were the dominant immunoglobulin class in these mucosal C. albicans infections. The IgA antibody titre may be of diagnostic value and seemed to be correlated to the severity of infections, with a higher level in oesophageal infections compared to oral infections. Antibody binding to these proteins was specific as the sera did not show the same enhanced recognition with bacterial or HeLa cell heat-shock proteins.
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Candida albicans exocellular antigens released into a synthetic culture medium: characterization and serological response in rabbits
More LessDifferent exocellular extracts were isolated by concentrating the supernatants of yeast- and mycelial-phase Candida albicans cultures incubated in a synthetic medium. The only difference between the extracts obtained from the two phases was the presence in those obtained from mycelial cultures of a polysaccharide-rich, high-molecular-mass component, migrating in SDS-polyacrylamide gels at a position that would correspond to proteins with molecular masses of 245–265 kDa. The electrophoretic band patterns obtained before and after concanavalin A-Sepharose 4B affinity column treatments confirmed that the 245–265 kDa band was the only one of mannoprotein nature. The extract obtained from 24 h mycelial-phase culture (EA) was selected as the exocellular antigen for this work. The dry weight of EA obtained from 1 litre of culture medium was 30 mg; it contained 53% carbohydrate (18·3% glucose and 21·7% mannose measured by gas-liquid chromatography) and 10% protein. Rabbit antisera against EA were absorbed with yeast-phase organisms and used to stain Western blots of gels loaded with EAs. These antisera clearly recognized bands in the 21, 33 and 44 kDa areas. The antiserum obtained was employed to develop a double-antibody enzyme-linked immunosorbent assay for measuring EA concentrations in a culture medium. Most of the EA was released during the exponential phase of growth.
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Macrophage chemiluminescence induced by interaction with transparent and opaque colonial variants of Mycobacterium intracellulare
More LessMacrophage (MΦ) chemiluminescence (CL) induced by interaction with the two types of colonial variants of Mycobacterium intracellulare was studied. A smooth, opaque and dome-shaped (SmD) colonial variant triggered more intense MΦ CL than did a smooth, transparent and flat colonial variant (SmT). MΦ CL-inducing activity of the SmD variant was reduced by heating or by treatments with either Pronase P, some endoglycosidases or Tween 80, thereby indicating that the SmD variant possesses MΦ CL-inducing substance(s) having peptide, sugar and/or lipid-like moieties. Treatment of the SmD variant organism with some endoglycosidases, such as cellulase, pectinase, dextranase or α-amylase decreased its MΦ CL-inducing ability. On the other hand, MΦ CL-inducing activity of the SmT variant was not affected by any of above treatments except that it was slightly increased by Pronase P treatment and reduced by α-amylase and dextranase.
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Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction
More LessSynthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibrio parahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibrio parahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio parahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibrio parahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. parahaemolyticus. This PCR protocol clearly identified TDH-producing strains of V. parahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.
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- Physiology And Growth
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Co-factor regeneration in the production of 1,2-epoxypropane by Mycobacterium strain E3: the role of storage material
More LessWhen grown on ethene Mycobacterium strain E3 produces epoxyalkanes from alkenes in an oxygen- and NADH-dependent reaction. The process of co-factor regeneration was studied by analysing the intracellular pools of NADH and storage material during the production of 1,2-epoxypropane from propene. With the depletion of NADH the production of 1,2-epoxypropane stopped. NADH could be regenerated from the oxidation of added co-substrate or from oxidation of storage material. Cells cultivated in chemostat culture under nitrogen limitation produced more 1,2-epoxypropane compared to cells cultivated under carbon limitation, due to their higher content of storage material. Addition of glucose to cells grown under carbon limitation stimulated the formation of 1,2-epoxypropane. The uptake of glucose resulted in the accumulation of storage material, which was utilized after depletion of the glucose. Glycogen and trehalose were the preferred forms of storage material used for co-factor regeneration. From the results it was concluded that formation and utilization of storage material play a crucial role in the process of co-factor regeneration in Mycobacterium strain E3.
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Thermosensitive cell growth mutants of Enterococcus hirae that elongate at non-permissive temperature are stimulated to divide by parental autolytic enzymes
More LessA series of thermosensitive cell growth mutants of Enterococcus hirae have been isolated. Most of these mutants elongate and some show reduced autolytic activity when incubated at the non-permissive temperature (42 °C) in comparison to the wild-type incubated at the same temperature. When mutants were incubated for longer than 15 min at 42 °C and were then shifted to 30 °C, a lag proportional to the time of preincubation at 42 °C was observed before division, indicating that a certain time is necessary to restore normal levels of an active molecule(s) needed for septum formation and division. The addition of wild-type muramidase-1 permitted the immediate formation of septa and a single cell division; further addition of the enzyme stimulated the cells to divide once again. The other E. hirae autolytic enzyme, peptidoglycan-hydrolase-2, which is found in the culture medium, seemed to be involved in separation of daughter cells but may also take over the function of muramidase-1. A key role of both enzymes in septum formation and division is postulated.
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Temperature-sensitive mutation in lytF, a new gene involved in autolysis of Escherichia coli
More LessA temperature-sensitive mutation in a new Escherichia coli gene, located at 62·5 min on the linkage map and designated lytF, resulted in bacteriolysis at the restrictive temperature. Temperature sensitivity and lytF-mediated lysis were simultaneously suppressed by either of two previously described unlinked mutations designated smhA1 and smhB1. The smhA1 and smhB1 alleles were originally isolated as specific extragenic suppressors of temperature-sensitive mutations in three other genes known as murH (99 min), lytD (13 min) and lytE (25 min) which conferred lysis phenotypes indistinguishable from that of the lytF mutation. The murH, lytD and lytE genes have been proposed to be related on the bases of phenotypic similarities and the specificities of their extragenic suppressors. It is now further proposed that lytF belongs to this group. The isolation of new alleles of smhA and smhB as extragenic suppressors of lytF further supports this proposal.
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Lysine secretion by wild-type Corynebacterium glutamicum triggered by dipeptide uptake
More LessIn Corynebacterium glutamicum peptide uptake increases the internal concentration of amino acids and thus triggers amino acid secretion. The peptide uptake system is stimulated by a factor of two in cells grown on pure peptone medium in comparison to peptone media with additional carbon sources. Uptake depends on the proton-motive force and shows a broad substrate spectrum. Peptide uptake is characterized by a K m of about 230 μm and a V max of 12 nmol min−1 (mg dry wt)−1 for the peptide lysyl-alanine (Lys-Ala). Lysine secretion in the wild-type of C. glutamicum does not show Michaelis-Menten-type kinetics as reported for the producing strains DG 52–5 and MH 20–22B. The secretion of lysine depends on the composition of the medium in which the cells were grown prior to the initiation of secretion by peptide uptake. The lack of secretion activity when the cells are shifted to peptone medium in the presence of chloramphenicol indicates that protein synthesis is necessary for this regulatory process.
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Utilization of organosulphur compounds by axenic and mixed cultures of Rhodococcus rhodochrous IGTS8
More LessGrowth assays reveal that Rhodococcus rhodochrous IGTS8 uses a wide range of organosulphur compounds as the sole source of sulphur, yet none of the compounds serve as carbon sources. Compounds that are utilized include thiophenes, sulphides, disulphides, mercaptans, sulphoxides, and sulphones. A convenient spectrophotometric assay (Gibbs assay), based on the chromogenic reaction of 2,6-dichloroquinone-4-chloroimide with aromatic hydroxyl groups, was developed and used in conjunction with GC/MS analyses to examine the kinetics of dibenzothiophene metabolism by axenic and mixed cell cultures of Rhodococcus rhodochrous IGTS8. The desulphurization trait is expressed at increasing levels during the exponential phase of growth and then declines in stationary-phase cells. Mixtures of streptomycin-resistant Rhodococcus rhodochrous IGTS8 and Enterobacter cloacae (an organism incapable of cleaving carbon-sulphur bonds in relevant test compounds) were prepared in ratios that varied over six orders of magnitude. Growth studies revealed that E. cloacae was able to gain access to sulphur liberated from organosulphur compounds by IGTS8; however, cell-to-cell contact appears to be required. These experiments also indicate that the desulphurization activity, on a per cell basis, is higher in mixed cultures than in axenic cultures.
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Characteristics of spores formed by surface and submerged cultures of Streptomyces albidoflavus SMF301
More LessStreptomyces albidoflavus SMF301 produced abundant spores in submerged cultures (submerged spores) as well as on solid media (aerial spores). The content of carbon, hydrogen, nitrogen, and phosphorus in submerged and aerial spores was similar; however, the contents of metal ions (K+, Na+, Ca2+ and Mg2+) were very different. Glutamic acid, alanine, and glycine, all known to be cell-wall components, were the major amino acids in both types of spores. However, cysteine was more abundant in submerged spores than in aerial spores. The major fatty acid in aerial spores was n-C18 (61·74%), whereas in submerged spores it was ai-C16 (33·68%). The contents of ai-C14, and ai-C17 in submerged spores were also very much higher than in aerial spores. Unsaturated fatty acids were found in both kinds of spores but not in mycelium; they were particularly abundant in submerged spores. The composition of menaquinones in the two kinds of spores also varied. The resistance of aerial spores to lysozyme digestion, mild acid treatment, heating and desiccation was higher than that of submerged spores, but the submerged spores were more resistant to sonication.
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Antimicrobial activity and biosynthesis of indole antibiotics produced by Xenorhabdus nematophilus
More LessWe have investigated the mechanism of action and physiology of production of the indole derivative antibiotics produced by the nematode-associated, entomopathogenic bacterium Xenorhabdus nematophilus. Maximum antibiotic concentration was reached during the late stationary phase of growth, and the antibiotic yield was appreciably enhanced by supplementation with tryptophan. Antibiotic biosynthesis apparently involved the removal of the side-chain carboxyl (C-1) carbon of tryptophan. The C-3 methylene carbon of tryptophan, on the other hand, was retained. The purified indole antibiotic was effective against both Gram-positive and Gram-negative bacteria at low to moderate concentrations causing a severe inhibition of RNA synthesis, accompanied by a less severe effect on protein synthesis. An isogenic pair of Escherichia coli strains differing at the relA locus was used to demonstrate that the swift reduction in total RNA synthesis is related to an antibiotic-induced accumulation of the regulatory nucleotide, ppGpp, in susceptible bacteria. The E. coli relA mutant, which does not exhibit any discernible increase in ppGpp upon antibiotic treatment, showed no decrease in growth or RNA synthesis. Using this antibiotic, it was also observed that ppGpp may be employed as a metabolic regulator in bacteria such as Pseudomonas putida, which have not previously been reported to employ ppGpp as a regulatory molecule. We propose that the indole derivative antibiotic exerts growth inhibitory control in susceptible bacteria by greatly enhancing synthesis of ppGpp, resulting in a rapid inhibition of RNA synthesis.
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Response of intracellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 and Rhizobium meliloti SU-47
More LessThe dynamic response of cellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 (0·25 m-NaCl) and Rhizobium meliloti SU-47 (0·4 m-NaCl) grown in NaCl-free medium was investigated in non-growing cell cultures and in cell suspensions, using in vivo NMR. After transferring cells grown in a NaCl-free medium to a glutamic-acid-free medium containing mannitol and NaCl, both strains immediately responded to the increased osmotic pressure by augmenting the cellular trehalose content of the cell. Without mannitol in the medium trehalose synthesis was slower, but clearly detectable. Its synthesis paralleled the breakdown of the reserve materials glycogen and poly-β-hydroxybutyric acid (PHB). NMR experiments with 25-fold-concentrated cell suspensions using 13C1-mannitol as substrate revealed that 15–20% of the trehalose synthesized was derived from mannitol, but 80–85% was from other sources. Trehalose was mainly formed from the internal pool of glycogen and/or PHB, whether mannitol was present or not, and reached 135 and 280 μg (mg cell protein)−1 in the strains TA-1 and SU-47, respectively. At low osmolarity, intracellular trehalose was metabolized by strains TA-1 and SU-47. Intracellularly accumulated phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of SU-47 cells grown in the absence of NaCl were neither degraded nor excreted after exposure to NaCl. Strain TA-1, which only makes neutral cyclic (1,2)-β-glucans, continued to synthesize and excrete cyclic (1,2)-β-glucans after exposure to NaCl. By using in vivo 31P-NMR, a sharp peak at 1·34 p.p.m. was present in cell suspensions of strain SU-47. This peak, representing glycerol-1-phosphate-substituted cyclic glucans, was absent in strain TA-1.
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- Plant-Microbe Interactions
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Changes in cultivar-specificity toward pea can result from transfer of plasmid RP4 and other incompatibility group P1 replicons to Pseudomonas syringae pv. pisi
More LessTransfer of RP4 and related replicons belonging to the Escherichia coli incompatibility group P (Pseudomonas aeruginosa IncP1) to races 2 and 6 of P. syringae pv. pisi was associated with the creation of two types of transconjugant, one resembling the parental race and the other showing an altered cultivar-specificity towards pea. The latter, irrespective of the parental race, exhibited a novel pattern of interaction with pea that corresponded to race 4; consequently such transconjugants were termed race 4-like. Curing of RP4 did not affect the phenotype, except in relation to the antibiotic resistances specified by RP4. The race 4-like strains were non-fluorescent when cultured on appropriate media (in contrast to the particular isolates of races 2 and 6 from which they were derived), showed an enhanced ability to inherit RP4 subsequently (at frequencies up to 10−1 per recipient) and differed from their parental race in their pattern of plasmid profile. The plasmid profiles were similar for all race 4-like strains irrespective of origin. There was no evidence that RP4 had recombined with DNA in the recipient and probing failed to detect the retention of any part of RP4 in cured strains. The inheritance of the related cosmid vector, pLAFR3, had similar effects in races 2 and 6. This observation is important since this vector has been widely used to clone avirulence genes in plant pathogenic bacteria. Transfer of the IncW plasmids S-a and R388 did not cause any changes in the fluorescence or cultivar-specificity of races 2 or 6. The novel avirulence expressed by the race 4-like variants derived from races 2 and 6 provides evidence for the presence in races 2 and 6 of an inhibitor/suppressor gene, which modulates the expression of the race 4-like avirulence gene.
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- Systematics
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Genetic structures of the B2 and B1 Escherichia coli strains responsible for extra-intestinal infections
More LessEscherichia coli strains causing human extra-intestinal infections may be divided into two groups, B1 and B2 according to the electrophoretic patterns of carboxylesterase B. This study compares the restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) for 45 B1 strains and 45 B2 strains to examine the genetic structure of B2 strains and to distinguish them from B1 strains. The isolates were chosen for diversity in their allozymes of esterases, B, A, C and I, their production of virulence factors (α-haemolysin, mannose resistant haemagglutinin and cytotoxic necrotizing factor) and certain O antigens, and their pathological and geographical origins. DNA was digested with HindIII and BamHI restriction enzymes and analysed by Southern blotting. The resulting rDNA RFLP patterns of B2 strains were distinct from those of the B1 strains. Moreover, the B2 strains appeared to be less heterogeneous than the B1 strains. The B2 strains gave 13 ribotypes (resulting from the combination of the rDNA RFLP patterns obtained with HindIII and BamHI digestions) while the B1 strains gave 32 ribotypes. Correspondence analysis of the data showed that several clusters of strains were identified in the B2 strains by particular ribotypes, certain associations of esterase B and A electrophoretic variants, O serotypes and virulence factor production. In contrast, these parameters appeared to be unrelated in the B1 strains, reflecting their heterogeneity. These findings, which differentiate two levels of genetic heterogeneity within E. coli pathogenic isolates, indicate that the B2 strains constitute a phylogenetically distinct group within the species.
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Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions
More LessTo develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15–1407 and B, 907–1407 (16S–16S); C, 1392–507 and D, 907–507 (16S–23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800–1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrn A-P) were demonstrated from 24 C. difficile strains ranging in size from 852–1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable in vivo, since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing C. difficile typing methods; it has universal applicability, it is objective and is moderately rapid.
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- Genome Analysis
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Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes
More LessPlasmid pEST4011 enables Pseudomonas putida PaW85 to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA). This new 2,4-D degradative plasmid has considerable homology with the regions of pJP4 containing the 2,4-D degradative genes (tfd). Restriction fragment BamHI-B of plasmid pEST4011, which has homology with this region, was cloned into the broad-host-range vector pKT240 and studied in P. putida PaW85. Restriction mapping, hybridization analysis and enzyme assays established the location of the genes for 2,4-D monooxygenase (tfdA), 2,4-dichlorophenol hydroxylase (tfdB), chlorocatechol 1,2-dioxygenase (tfdC) and the tfdR and tfdS regulatory genes on this fragment. Plasmid pEST4012 is a derivative of pEST4011 derived through the spontaneous deletion of a 42 kbp DNA fragment, which results in the loss of the 2,4-D+ and 3-CBA+ phenotype. We present here the physical maps of pEST4011 and pEST4012. In spite of the similarities in functions, the size (70 kbp), order of catabolic genes and restriction pattern of pEST4011 are clearly different from those of pJP4.
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Volumes and issues
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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)