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Volume 131,
Issue 7,
1985
Volume 131, Issue 7, 1985
- Biochemistry
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The Uptake and Production of Molecular Hydrogen by Unicellular Cyanobacteria
More LessSummary: Nine strains of unicellular non-nitrogen-fixing cyanobacteria had high rates of hydrogen evolution and uptake compared with filamentous nitrogen-fixing strains. Maximal hydrogen uptake activities were observed in assays including oxygen under both light and dark conditions suggesting the participation of the ‘Knallgas’ reaction. Nitrogenase activity, measured as acetylene reduction, was not found in aerobic cultures of any of the unicellular strains tested, and the capacity to synthesize nitrogenase under anaerobic conditions was absent in all five of the strains tested under these conditions. Thus the capacity to synthesize nitrogenase is not a prerequisite for hydrogenase synthesis and hydrogen evolution by these unicellular cyanobacteria. Rates of hydrogen evolution and uptake by six of the unicellular strains were increased following adaptation with CO2/H2 gas mixtures.
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Glycerol Oxidation and Triose Reduction by Pyridine Nucleotide-linked Enzymes in the Fission Yeast Schizosaccharomyces pombe
More LessSummary: The fission yeast Schizosaccharomyces pombe has been shown to produce four separate pyridine nucleotide-linked glycerol dehydrogenases (or triose reductases), distinguished by differences in their coenzyme specificity (NAD+ or NADP+) and oxidation product (dihydroxyacetone or glyceraldehyde). Evidence for four separate activities was obtained by heat inactivation studies, comparison of cells grown under different conditions, and separation and partial purification of the enzymes. One enzyme, a glycerol: NAD+ 2-oxidoreductase is repressed by glucose but not induced by glycerol and appears to function primarily in glycerol catabolism. The second, a glycerol: NADP+ 2-oxidoreductase is stimulated by growth on glucose and appears to function as a dihydroxyacetone reductase involved in glycerol synthesis. The third has the properties of a glycerol: NADPT+ oxidoreductase, while the fourth is, in fact, alcohol dehydrogenase (alcohol: NAD+ oxidoreductase) which possesses weak activity as a glycerol: NAD+ 1-oxidoreductase.
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Purification and Properties of Glycerol: NAD+ 2-Oxidoreductase (Glycerol Dehydrogenase) from Schizosaccharomyces pombe
More LessSummary: Glycerol: NAD+ 2-oxidoreductase (glycerol dehydrogenase, EC 1.1.1.6) from Schizosaccharomyces pombe has been purified to homogeneity. The protein has a molecular weight of about 400000; it can be dissociated into identical subunits of molecular weight 47000, and is probably an octamer. The pH optimum for glycerol oxidation is 10·0 or higher and for the reverse reaction is 6·0. Oxidation occurs specifically at C2 of glycerol to produce dihydroxyacetone and not glyceraldehyde. Several diols with hydroxyls on adjacent carbon atoms can be oxidized and corresponding carbonyl compounds reduced, 1,2-propanediol being oxidized 1·6 times more rapidly than glycerol. The forward reaction has a specific requirement for NAD+ as coenzyme whereas NADPH shows about one-third of the activity of NADH for the reverse reaction. The monovalent cations K+ and NH+ 4 activate the enzyme, while Na+ and Li+ counteract this effect. Some thiol and chelating agents are inhibitory while thiol antagonists, Mn2+, and to a lesser extent Zn2+, stimulate activity. Apparent K m and V max values have been determined. The enzyme is similar to but not identical with the glycerol dehydrogenases isolated from Escherichia coli and Klebsiella aerogenes (K. pneumoniae).
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Effect of Allylamine Antimycotic Agents on Fungal Sterol Biosynthesis Measured by Sterol Side-chain Methylation
More LessSummary: Sterol side-chain (C-24) methylation was assayed by incorporation of radioactivity from [Me-14C]methionine into the ergosterol fraction in cells of the pathogenic fungi Candida albicans, Candida parapsilosis and Trichophyton mentagrophytes. Methylation at C-24 occurred after nuclear demethylation in all cases. The method was used to measure ergosterol biosynthesis inhibition by the allylamine antimycotics naftifine and SF 86-327, which are known to block squalene epoxidation. In C. albicans cells treated with SF 86-327 (1 mg 1−1) to fully inhibit squalene epoxidation, C-24 methylation continued for several hours at about 40% of the control rate. This residual biosynthesis was probably due to methylation of endogenous sterol precursors. The degree of residual biosynthesis in the three fungi correlated well with their susceptibility to SF 86-327. The highly susceptible dermatophyte T. mentagrophytes had negligible residual sterol biosynthesis. These differences were not due to inhibition of methionine uptake. For naftifine (100 mg 1−1) there was evidence of a second inhibitory action C. albicans. A cell-free assay indicated that this was due to direct inhibition of the C-24 methyltransferase.
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Acriflavine-binding Capacity Controlled by the acrA Gene of Escherichia coli
H. Nakamura and T. ShinyaSummary: The acrA mutation in Escherichia coli led to a substantial increase of the acriflavine-binding capacity of the cell, whereas the related mutations acrB (gyrB) and acrC did not. Metal ions such as Na+, K+, Mg2+, Ca2+and Al3+effectively released the bound acriflavine, in proportion to their ionic strengths. The presence of cations, in fact, increased the survival fraction of the cells in the acriflavine-containing medium. Polymyxin B, an antibiotic which binds to membrane phospholipid, competed with acriflavine for binding sites. Cell wall digestion by treatment with lysozyme and EDTA slightly decreased the acriflavine-binding capacity. Almost no difference was observed in acriflavine-binding capacity between intact cells and cells from which lipopolysaccharide has been extracted (46·9% removed from the acrA cells and 47·4% from the acrA +cells). Acriflavine bound to the cells was most effectively extracted by ethanol containing 1% HCl or by 2% (w/v) SDS. The difference in the acriflavine-binding capacity between the acrA and acrA +cells was also observed in the spheroplasts. These facts indicate a relationship between the acrA gene product and the acriflavine-binding capacity of the cells.
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Nitrogenase of Sesbania Rhizobium Strain ORS571: Purification, Properties and ‘Switch-off’ by Ammonia
More LessSummary: Nitrogenase from the Rhizobium strain ORS571, which forms both root and stem nodules on the tropical plant Sesbania rostrata, was purified from free-living diazotrophically grown organisms. The enzyme complex was resolved into two protein components resembling those obtained from other diazotrophs. Both components were purified to homogeneity as judged by SDS-gel electrophoresis. Component 1, a Mo-Fe protein, had a M r of 219000 and contained 1·2 g-atoms Mo mol−1, 22·5 g-atoms Fe mol−1and 20·5 g-atoms acid-labile S mol−1. It consisted of two types of subunit of M r 56000 and 59000. The specific activity [nmol product formed min−1(mg protein)−1] of component 1, when assayed in the presence of an optimum concentration of component 2 (molar ratio 1:40), was 1250 for acetylene reduction and 1090 for hydrogen evolution. Component 2, an Fe protein, had a M r of 74000 and contained 3·1 g-atoms Fe mol−1and 3·1 g-atoms acid-labile S mol−1. It consisted of a single type of subunit of M r 36000. The specific activity of component 2, when assayed in the presence of an optimum concentration of component 1 (molar ratio 1:1) was 1700 for acetylene reduction and 1305 for hydrogen evolution. Nitrogenase activity of strain ORS571 was subject to ‘switch-off’ when ammonia was added to a N2-fixing culture. This effect was independent of protein synthesis and was reversible. Nitrogenase components 1 and 2 isolated from ‘switched-off’ organisms were purified to homogeneity. Both components had the same mobility on SDS-gels as those isolated from active cultures. ‘Switch-off’ resulted in a decrease in the specific activity of component 2 from 1700 to 280. Component 1 remained fully active. The addition of Mn2+to a crude extract containing inactivated nitrogenase did not restore activity.
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The Catabolism of 4-Hydroxyacetophenone by an Alcaligenes sp.
More LessSummary: A bacterium capable of growth on 4-hydroxyacetophenone was isolated from soil and identified as an Alcaligenes sp. Intact cells rapidly oxidized (4-hydroxybenzoyl)methanol, 4-hydroxybenzoate and protocatechuate as well as the growth substrate, and also converted the substrate analogue (4-methoxybenzoyl)methanol to 4-methoxybenzoic acid. When provided with NADH, cell-free extracts oxidized 4-hydroxyacetophenone to 4-hydroxybenzoate and formate, the same products as were formed from (4-hydroxybenzoyl)methanol without NADH. The oxidation of 4-hydroxybenzoate by cell-free extracts required NADPH and the product from both this and protocatechuate oxidation was 3-oxoadipate. A pathway for the catabolism of 4-hydroxyacetophenone, by hydroxylation to (4-hydroxybenzoyl)methanol followed by oxidative cleavage to 4-hydroxybenzoate and formate and hydroxylation of the 4-hydroxybenzoate to protocatechuate, is proposed. Oxidation of protocatechuate was by the ortho pathway. The key enzymes in the proposed pathway were induced by growth on 4-hydroxyacetophenone.
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- Development And Structure
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Gametogenesis in Liquid Cultures of Chlamydomonas eugametos
More LessSummary: At the end of the exponential phase of vegetative growth in liquid cultures, cells of Chlamydomonas eugametos develop into gametes. Gametogenesis can occur without nitrogen deficiency. In continuous light, mating competence lasts only for a few hours, resulting in a low percentage of gametes. Nevertheless, mating competence is considered to be a general property of newly born cells in late exponential phase, since cultivation of both mating-types in mixed cultures normally leads to a high yield of paired cells. No evidence was found for an influence of the presence of partner gametes on gametogenesis. During gametogenesis of the mt − mating-type, extractable biologically active agglutination factor appears in the cell body. Subsequently, after a gametogenic division, mating competence and agglutinability are expressed, both fluctuating synchronously with the light/dark regime.
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Reversal of Multicellular-form Development in a Conditional Morphological Mutant of the Fungus Wangiella dermatitidis
More LessSummary: Yeast cells of the temperature-sensitive morphological mutant strain Mc3 of the polymorphic fungus Wangiella dermatitidis were allowed to initiate multicellular-form development at the restrictive temperature (37 °C), then induced to resume budding yeast growth by downshift to the permissive temperature (25 °C). The kinetics of the resumption of yeast budding were investigated, and the dependence of bud initiation upon DNA synthesis, protein synthesis and microtubule function was probed. The results indicated that the thermolabile event in strain Mc3 was a cell cycle event involved in yeast budding, and that developing multicellular forms retained most of the machinery necessary to reinitiate budding. However, initiation of budding required the synthesis of protein, and possibly RNA, suggesting that one or more proteins involved in initiation of budding were not present in the developing multicellular forms.
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- Ecology
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Degradation of Fungal and Actinomycete Mycelia by Agaricus bisporus
More LessSummary: Commercial wheat straw/horse manure compost used for production of the cultivated mushroom Agaricus bisporus contains a fungal and actinomycete microbial biomass of some 1.5% before inoculation with A. bisporus. It has been shown that these mycelia can be used by A. bisporus as a sole source of carbon, nitrogen and phosphorus. Mycelium of the thermophilic fungus Humicola insolens var. thermoidea, a major component of compost microbial biomass, was digested over a 28 d period by A. bisporus growing in liquid culture, as determined by light and electron microscopy. Mycelial walls of H. insolens var. thermoidea contained (by wt) 30% glucose, 22% glucosamine, 6-8% galactose, 2-3% mannose and traces of other sugars. Enzymes that possibly degrade fungal and actinomycete cell walls were produced by A. bisporus during growth on such mycelia, and activities of other extracellular enzymes involved in the decomposition of cytoplasmic contents were determined.
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- Genetics And Molecular Biology
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Inverse Relationship between Formation of Phenotypically Recombinant Clones and Cell Wall Regeneration after Fusion of Bacillus subtilis Protoplasts
More LessSummary: The prototrophic clones formed after fusion of mixed protoplasts from two polyauxotrophic strains of Bacillus subtilis have been counted both as L-form colonies and as bacterial colonies, by plating on selective medium in the presence and in the absence of methicillin. On average, one hundred times as many prototrophic colonies were counted when cell wall regeneration was prevented by the antibiotic. Thus genetic inactivation, which occurs regularly in bacterial exfusants, may be dependent on cell wall regeneration.
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Fosfomycin-resistance Plasmids Determine an Intracellular Modification of Fosfomycin
More LessSummary: Escherichia coli cells carrying fosfomycin-resistance plasmids modify fosfomycin intracellularly. The product of this modification (fosfomycin-derivative) differs from fosfomycin in chromatographic mobility, but the chemical nature of the modification is not yet known. Fosfomycin-derivative appears to have a cytoplasmic location and lacks antibiotic activity. The modification system can be saturated by an excess of fosfomycin in the incubation media.
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Cloning of a DNA Fragment from Streptomyces griseus which Directs Streptomycin Phosphotransferase Activity
More LessSummary: DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into Bg/II-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4·45 and 11·55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1·95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1·5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.
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Isolation of Cyanidium caldarium and Porphyridium purpureum DNA Fragments Capable of Autonomous Replication in Yeast
More LessSummary: DNA fragments from the unicellular red algae Cyanidium caldarium and Porphyridium purpureum were inserted at various sites of plasmid pLG4 (pBR325 + a HindIII fragment bearing the yeast arg4 gene) with the purpose of isolating sequences supporting autonomous replication of plasmids in Saccharomyces cerevisiae. Plasmid pools were prepared in Escherichia coli then used to transform the arg4 yeast strain X3656-7 D to prototrophy. The presence of free plasmids in the yeast transformants was demonstrated by Southern blotting hybridization between yeast DNA and 32P-labelled pBR325 and by the transformation of E. coli argH with DNA from yeast transformants. Hybrid plasmids recovered from Arg+bacterial transformants transformed yeast at high frequency. They contained Aval fragments of C. caldarium total DNA, EcoRI fragments of P. purpureum satellite DNA and one BamHI fragment of P. purpureum main DNA. These new plasmids have unique restriction sites which make them convenient vectors for cloning in yeast and possibly in algae and other plants.
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Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis
More LessSummary: We have constructed secretion vector plasmids that have unique Bg/II sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exolarge and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.
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Reiteration of Nitrogen Fixation Gene Sequences and Specificity of Rhizobium in Nodulation and Nitrogen Fixation in Phaseolus vulgaris
More LessSummary: We have previously reported that Rhizobium phaseoli has multiple copies of nitrogen-fixation gene sequences. In this work we extend our analysis to cover a broader range of R. phaseoli strains and other rhizobia isolated from different legumes. Our results indicate that most R. phaseoli strains have reiterated nifH sequences. Reiterations are also found in rhizobia from nodules of other Phaseolus species and from the close relative of Phaseolus, Pachyrhizus. However, nifH gene reiteration is not always found in the rhizobia able to nodulate and fix nitrogen in Phaseolus, since some strains isolated from nodules of Phaseolus vulgaris as well as from other legumes are able to establish an effective symbiosis with Phaseolus vulgaris and do not show reiterations. We propose that there are different evolutionary lines of R. phaseoli. The reiteration of nif genes may be considered a marker for the most abundant and specialized of these evolutionary lines.
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The Use of Cloned nif Regulatory Elements from Klebsiella pneumoniae to Examine nif Regulation in Azotobacter vinelandii
More LessSummary: Regulatory genes controlling nif expression and also nif promoters fused to lacZ from Klebsiella pneumoniae were cloned on wide-host-range plasmids and introduced into Azotobacter vinelandii to compare regulation of nif expression in the two organisms. A low-copy-number plasmid carrying K. pneumoniae nif A corrected an A. vinelandii Nif−regulatory mutation, whereas a plasmid carrying ntrC did not. A high-copy-number plasmid carrying K. pneumoniae nifL eliminated nif expression in K. pneumoniae but not in A. vinelandii. K. pneumoniae nifL- and nifF-lacZ fusions were expressed strongly in A. vinelandii and were not repressed by ammonium. A nifH-lacZ fusion was not expressed in any conditions in this background except very weakly when K. pneumoniae nifA was also present. The implications of these findings are discussed.
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Molecular Cloning of WHI2, a Gene Involved in the Regulation of Cell Proliferation in Saccharomyces cerevisiae
More LessSummary: The WH12 gene of Saccharomyces cerevisiae plays a key role in co-ordinating cell proliferation and nutrient availability. A 2·6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.
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Evidence for Related Virulence Sequences in Plasmids of Salmonella dublin and Salmonella typhimurium
More LessSummary: Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1·5 kbp from the TnA insertion in M51 and 6·5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.
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- Pathogenicity And Medical Microbiology
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The Purification and Some Properties of H-lysin from Aeromonas salmonicida
More LessSummary: H-lysin from Aeromonas salmonicida has been purified 1770-fold by freeze fractionation, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The purified material was predominantly H-lysin, devoid of detectable T-lysin, caseinase or gelatinase activity, although glycerophospholipid: cholesterol acyltransferase (GCAT) activity was present. The results suggested that H-lysin and GCAT activities were due to different extracellular products. Studies of the kinetics of haemolysis indicated that the H-lysin had an enzymic mode of action, and that initial erythrocyte damage appeared to precede lysis of the cell. The H-lysin was lethal to cultured rainbow trout gonad cells and leucocytes, but when it was injected intravenously in rainbow trout no pathological effects were observed.
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