- Volume 130, Issue 7, 1984
Volume 130, Issue 7, 1984
- Biochemistry
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Properties and Role of Sulphite: Cytochrome c Oxidoreductase Purified from Thiobacillus versutus (A2)
More LessSulphite: cytochrome c oxidoreductase (sulphite dehydrogenase) was purified 2000-fold from Thiobacillus versutus (A2). The enzyme monomer had a molecular weight of 44000 and a pI value of 4·5 α 0·3. Cytochrome c 551 was intimately associated with the enzyme: separation of the two greatly decreased sulphite dehydrogenase activity, which was not restored by remixing them. The enzyme had a pH optimum around pH 8·0, exhibited a K m of 14 µm for sulphite, and was inhibited noncompetitively by phosphate, with a K i value of 12 mm. It was also inhibited by p-hydroxymercuribenzoate and cyanide. Its involvement in the oxidation of thiosulphate in T. versutus is discussed.
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Effect of Nitrogen Source on Lipid Accumulation in Oleaginous Yeasts
More LessThe effect of various nitrogen sources on lipid accumulation by 17 species and strains of yeast was examined. Organic nitrogen sources resulted in considerably increased lipid contents only in strains of Rhodosporidium toruloides. Lipid accumulation in Rs. toruloides CBS 14 increased from 18% (w/w), with NH4Cl as nitrogen source, to above 50% (w/w) when glutamate, urea or arginine was used. Stimulation of lipid production by glutamate was not observed when the yeast was grown in continuous culture with nitrogen-limiting medium. The increase in lipid content of glutamate-grown cells in batch culture was accompanied by a marked increase in the intracellular citrate concentration and its excretion from the cells. The pattern of citrate accumulation in glutamate-grown cells was mirrored by the accumulation of other metabolites, especially 2-oxoglutarate and NH+ 4 ions, which were produced as a result of the increased catabolism of glutamate. It is proposed that the products of glutamate metabolism in Rs. toruloides play a major role in regulating the flux of carbon to precursors of lipid biosynthesis, such as citrate.
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Influence of Nitrogen Metabolism on Lipid Accumulation by Rhodosporidium toruloides CBS 14
More LessRhodosporidium toruloides CBS 14 was grown in batch culture with urea as principal nitrogen source. The lipid content increased from 18% (w/w) with NH+ 4-grown cells to 52% (w/w) after 90 h growth. Urea was rapidly taken up and catabolized to release intracellular NH+ 4, which accumulated between 10 and 30 h growth. The increase in pool NH+ 4 content was mirrored by an increase in citric acid accumulation and excretion from the cells. The production of intracellular NH+ 4, sufficient to permit lipid accumulation, could be attributed to the increase in activity of urease over this period. Similarly, other catabolic enzymes, such as arginase, threonine dehydratase and NAD+: glutamate dehydrogenase, were also induced (or derepressed) when the respective amino acids were used as medium nitrogen source. Growth with mixed organic and inorganic nitrogen compounds considerably decreased the lipid content and was accompanied by a reduction in activity of the various catabolic enzymes concerned. The significance of nitrogen catabolism during lipid accumulation in this yeast is discussed.
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Cyclic GMP and Cyclic AMP Binding Proteins in Myxococcus xanthus
More LessThree binding proteins, one specific for guanosine 3′: 5′-monophosphate (cGMP) and two specific for adenosine 3′: 5′-monophosphate (CAMP) have been partially purified from vegetative cells of Myxococcus xanthus M300. The cGMP binding activity was found in the periplasmic shock fluid. Scatchard analysis indicated only a single class of binding sites with high affinity (apparent K D, 42 nm). The two cAMP binding activities were physically distinct, as indicated by their elution patterns from DEAE-cellulose, K D values and cellular locations. The cytoplasmic cAMP binding protein, which is probably identical to that previously isolated from developing myxospores of M. xanthus had an apparent K D of 57 nm, whereas the periplasmic cAMP binding protein had an apparent KD of 1 μm. During development, the nucleotide binding proteins exhibited changes in activities consistent with their postulated roles during fruiting body development.
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Adenylate Cyclase and Guanylate Cyclase in Myxococcus xanthus
More LessMyxococcus xanthus M300 vegetative cells contained significant amounts of adenylate and guanylate cyclase activity. The latter was distributed between the 100000 g supernatant and pellet fractions, required divalent cations for activity and exhibited an apparent K m of 1 mm. Adenylate cyclase activity was detected both in the l00000 g supernatant and pellet. The supernatant enzyme had an apparentK m of 220 μm with a Hill coefficient of 1·9, whereas the pellet enzyme had a K m of 72 μm and a Hill coefficient of 1·0. The isoenzymes differed in their pH optima and divalent cation requirements for optimal activity. During development of Myxococcus xanthus, the nucleotide cyclase activities exhibited changes that were substantially consistent with the roles postulated for each in a previously proposed model.
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The Detection and Analysis of Chitinase Activity from the Yeast Form of Candida albicans
More LessChitinase activity was detected in the supernatant fraction of a high-speed centrifugation preparation of broken Candida albicans yeast cells. The enzyme showed peak activity during the rapid budding phase of growth and was found to parallel the chitin synthase activity. The optimum conditions for the hydrolysis of chitin, regenerated from acetylation of chitosan, were determined. Analysis of the kinetics of the enzyme-substrate interaction and a measurement of their binding suggests that an equilibrium binding situation exists and that the kinetics follow a Langmuir isotherm interaction.
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- Development And Structure
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Shape of Nascent and Completed Poles of Bacillus subtilis
More LessThe zonal dome model has previously been shown to fit the shape of the poles of the Gram-positive Streptococcus faecium quite accurately, but measurements of the angle that the pole of Bacillus subtilis makes with the cylinder portion of the cell show that the poles of this organism do not fit this model. Furthermore, the diameters of curvatures in different parts of the completed and nascent pole are contrary to the predictions of the zonal dome model and several other proposed models. The results suggest that the poles of Gram-positive rods are generated by a mechanism designated the ‘split-and-stretch’ model. The model assumes that no additional murein is inserted as the septal wall is split and externalized.
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Glutamine Requirement for Aerial Mycelium Growth in Neurospora crassa
More LessFive amino acids are accumulated during vegetative growth of Neurospora crassa, particularly during the prestationary growth phase. Alanine, glutamine, glutamate, arginine and ornithine comprised over 80% of the total amino acid pool in the mycelium. Amino acid pools of different amino acid auxotrophs were followed during the partial transformation of a mycelial mat into an aerial mycelium. The mycelial mat under starvation and in direct contact with air rapidly formed aerial mycelium, which produced thereafter a burst of conidia. During this process, glutamine and alanine in the mycelial mat were consumed more rapidly than other amino acids; in the growing aerial mycelium, glutamate and glutamine were particularly accumulated. Of the amino acids that were initially accumulated in the mycelial mat, only a high glutamine pool was required for aerial mycelium growth induced by starvation. This requirement for glutamine could not be satisfied by a mixture of the amino compounds that are synthesized via glutamine amidotransferase reactions. It is proposed that glutamine serves as a nitrogen carrier from the mycelial mat to the growing aerial mycelium.
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Glutamine Metabolism During Aerial Mycelium Growth of Neurospora crassa
More LessDuring vegetative growth, glutamine is accumulated in the mycelium of Neurospora crassa. This high pool of glutamine seems to be required for aerial mycelium growth. Enzymes responsible for the synthesis and catabolism of glutamine were measured before and during the partial transformation of a mycelial mat into aerial mycelium. In the transforming mycelial mat, considerable activities of the biosynthetic NADP-glutamate dehydrogenase and glutamine synthetase (predominantly β polypeptide) and also some activity of glutamate synthase were observed. In the aerial mycelium, glutamine synthetase (predominantly β polypeptide) was detected, but very low activities of NADP-glutamate dehydrogenase and glutamate synthase were observed, indicating that nitrogen can be assimilated in the mycelial mat but hardly in the aerial hyphae. Accumulated glutamate in the aerial mycelium could derive from glutamine. No glutaminase activity could be detected. It is suggested that glutamate is formed through the activities of the glutamine transaminase-ω-amidase pathway and another transaminase. High activities of glutamine and alanine transaminases were observed in the aerial mycelium. These results are discussed in terms of the possible role of glutamine as a nitrogen carrier from the mycelium to the growing aerial hyphae.
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- Genetics And Molecular Biology
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The Genomes of Desulfovibrio gigas and D. vulgaris
More LessTwo-dimensional electrophoresis of sequential double-restriction digests showed that the genome of Desulfovibrio gigas comprised 1·63 × 106bp (1·09 × 109 Dal) of DNA; an ammonia-limited chemostat population possessed an average of nine genomes per cell and a multiplying batch culture possessed ∼17 genomes per cell. The genome size of D. vulgaris (Hildenborough) was 1·72 × 106 bp (1·14 × 109 Dal); a population from an ammonia-limited batch culture contained four genomes per cell. Control digestions and analyses with Escherichia coli GM4 agreed reasonably with published values: a genome size of 3·95 × 106 bp and approximately two genomes per cell from a stationary batch culture in glucose minimal medium. Desulfovibrio gigas carried two plasmids of ∼70 MDal (1·05 × 105 bp) and ∼40 MDal (6 × 104 bp); D. vulgaris (Hildenborough) contained one of ∼130 MDal (1·95 × 105 bp). Single plasmids were also detected in a second strain of D. vulgaris and in strain Berre sol of D. desulfuricans but not in 10 other desulfovibrios including representatives of D. desulfuricans, D. vulgaris, D. salexigens and D. africanus.
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Genome Size and Complexity in A◄otobacter chroococcum
All of eight strains of Azotobacter chroococcum examined contained between two and six plasmids ranging from 7 to > 200 MDal in size. Strain MCC-1, a derivative of NCIMB 8003, was cured of various of the four largest of its five plasmids and the phenotypes of the strains compared. All fixed nitrogen and exhibited uptake hydrogenase activity. No differences were observed in carbon source utilization or antibiotic, heavy metal or UV resistance. The genome sizes of two strains of A. chroococcum were determined by two-dimensional electrophoresis. Strain CW8, an isolate from local soil containing two small plasmids of 6 and 6·5 MDal contained unique DNA sequences equivalent to 1·78 × 106 (±20%) bp (1·2 × 109 Dal). In strain MCD-1, a derivative of MCC-1, containing a 190 MDal and 7 MDal plasmid, the genome size was 1·94 × 106 (±20%) bp. In exponential batch cultures, both contained 20 to 25 genome equivalents per cell. MCD-1 exhibited complex UV kill kinetics with a marked plateau of resistance; CW8 showed a simple response inconsistent with the possibility of organization of its DNA into identical chromosome copies capable of independent segregation.
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Gene Amplification in Bacillus subtilis
More LessA strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 µg chloramphenicol ml−1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in R factor transitioning. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.
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Plasmids of the Epiphytic Bacterium Erwinia uredovora
More LessTwo Erwinia uredovora strains, 20D3 and 20D31, were analysed for the presence of plasmids. Agarose gel electrophoresis of crude lysates of these strains indicated that 20D3 carried three plasmids with molecular sizes of 88 kb, 216 kb and 260 kb while 20D31 contained five plasmids with molecular sizes of 7·8 kb, 28 kb, 105 kb, 180 kb and 260 kb. Strain 20D31 proved to be sensitive to phage GU5, suggesting that at least one of its plasmids coded for a derepressed transfer system. Curing and mobilization experiments indicated that the GU5 sensitivity was determined by the 28 kb plasmid, which was conjugative and belonged to the IncN group. Both strains lost yellow pigmentation and thiamin prototrophy when grown at elevated temperature (39 °C) or in the presence of nalidixic acid or SDS. The irreversible loss of both traits was ascribed to the curing of the 260 kb plasmid. Yellow pigmentation was due to the production of carotenoids.
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Erwinia uredovora 20D3 Contains an IncM Plasmid
More LessErwinia uredovora was lysogenized with phages Mucts62 and D108-1, a mini derivative of D108cts10 carrying a Cmr marker. The 20D3(Mucts62)(D 108-1) lysogen was induced and used as donor in a mating with a phage-resistant Escherichia coli; the Cmr marker was transferred to this recipient strain at low frequency. The analysis of the Cmr transconjugants indicated that they had acquired the pULG3 plasmid of E. uredovora associated with a D108-1 prophage. Subsequent transfer of these pULG3 derivatives within E. coli indicated that pULG3 was conjugative. Incompatibility experiments showed that pULG3 is a member of the IncM group.
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Genetic Evidence for the Direction of Transcription of the trfA Gene of Broad Host Range Plasmid RK2
More LessFor replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12·0 and 12·7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16·0 and 17·4 kb, which provides a trans-acting product necessary for oriV RK2 function. The properties of an insertion mutant of a mini-RK2/ColEl hybrid plasmid suggest that the trfA promoter lies clockwise from the 17·4 kb RK2 coordinate. Fusion of the trfA gene lacking its normal promoter to the E. coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2. Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression. The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54·0 and 56·0 kb on the RK2 genome.
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Molecular Genetic Analysis of the trfB and korB Region of Broad Host Range Plasmid RK2
More LessA 3·2 kb region of the broad host range IncP plasmid RK2 (indistinguishable from RP1, RP4, R68 and R18) anticlockwise from the EcoRI site may be separated phenotypically into three loci. The trfB/korA/korD locus both complements a temperature-sensitive maintenance mutation and suppresses the deleterious effects of the loci kilA and kilD; the incC locus expresses incompatibility towards complete RK2-like replicons, and the korB locus suppresses the host lethal effect of the kilB locus. Transcriptional fusions of the galK gene to various segments of this region revealed that all three loci are transcribed anticlockwise from a common promoter. A weak secondary promoter may also contribute to the expression of korB. Analysis of the sizes of the polypeptides produced from these segments led to the identification of two cistrons, the first encoding a polypeptide of 38 kDal associated with incC function and the second a polypeptide of 49 kDal associated with korB activity. The trfB/korA/korD activities are associated with a polypeptide of 14 kDal which may be an N-terminal fragment of the incC-associated polypeptide.
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Characterization of High-level Aminoglycoside Resistance in a Strain of Streptococcus pneumoniae
More LessThe aminoglycoside phosphotransferase (APH)(3′)(5″)-III has been characterized from Streptococcus pneumoniae BM4200, which is resistant to high levels of aminoglycosides. The phosphotransferase was apparently chromosomally-encoded and was responsible for the high-level resistance. The enzyme was not notably pH-dependent, was heterogeneous after isoelectric focusing, with pI values of approximately 4·8 and 5·1, and had an apparent molecular weight of 32500 after SDS-PAGE.
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Detection of a Membrane-associated Protein on Detached Membrane Ribosomes in Staphylococcus aureus
More LessMembrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71000, 46000 and 41000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71000, 60000, 46000 and 41000 seemed to be present in stoichiometrically equivalent amounts in the complex.
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The Effect of Plasmid R391 and Other IncJ Plasmids on the Survival of Escherichia coli After UV Irradiation
More LessThe presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA +cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that postirradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.
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- Pathogenicity And Medical Microbiology
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Purificaiton by Surcrose Density Gradient Zonal Centrifugatin and Affinity Coloum Chromatogrphy of Antigenic Ssubstancaes of Antigenic Susbstances from the Livers and Mice Infected with Tyzzer’s Diseae
More LessAntigenic substances from livers of mice infected with Tyzzer's disease were purified by means of sucrose density gradient zonal centrifugation and affinity column chromatography using antiserum and checking antigenicity with the complement fixation test. Fractions obtained from zonal centrifugation fell into three main groups with different molecular weights, two of which (Fr. I and Fr. 11) positively reacted with antiserum in the complement fixation test. Both fractions were further purified by affinity column chromatography. The molecular weights of the main antigenic substances derived from Fr. I and Fr. I1 were determined to be about 52000 and 66000, respectively, by means of SDS-PAGE.
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