- Volume 129, Issue 6, 1983
Volume 129, Issue 6, 1983
- Biochemistry
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Steroids from the Myxobacterium Nannocystis exedens
More LessSqualene and three sterols were isolated from Nannocystis exedens (Myxobacterales). The sterol content was about 0·4% of the dry weight; squalene content was in the same range. The main component was identified as cholest-8(9)-en-3β-ol. [14CMevalonate was readily incorporated into the sterols. The bacterium was not inhibited by nystatin or amphotericin B.
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Purification and Properties of Two Neutral Proteinases from Aspergillus nidulans
More LessTwo proteinases have been purified from mycelial extracts of Aspergillus nidulans. Both enzymes have pH optima between 6·5 and 7·5 and are inhibited by phenylmethane sulphonyl fluoride and by di-isopropyl fluorophosphate. The molecular weights and isoelectric points of proteinase I and proteinase II are 30900 and 30000, and 4·6 and 4·3, respectively. Both enzymes have a similar range of substrate specificities. The principal differences in their properties are that proteinase I is sensitive to inhibition by 1 mM mercurials whereas proteinase II is not appreciably inhibited at this concentration, and that proteinase I is much more sensitive to denaturation by urea, guanidine hydrochloride and sodium dodecyl sulphate.
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The Soluble Cytochrome Oxidase of Nitrosomonas europaea
More LessThe soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be a copper protein devoid of haem, not a cytochrome o as was previously assumed. The native molecular weight was 120000 and the subunit molecular weight 35000. Soluble cytochrome oxidase activity co-purified with nitrite reductase activity; both activities were almost certainly associated with the same protein. The possible physiological role of the nitrite reductase activity is discussed.
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The Electron Transport Chain of Escherichia coli Grown Anaerobically with Fumarate as Terminal Electron Acceptor: an Electron Paramagnetic Resonance Study
More LessThe electron transport chain of Escherichia coli grown anaerobically on glycerol with fumarate as terminal electron acceptor, has been studied using electron paramagnetic resonance (EPR) spectroscopy. The analysis did not include cytochromes, but was confined to the lower potential (dehydrogenase) section of the electron transport chain. Several ferredoxin-type centres were detected and partially characterized, and the possible presence of iron-sulphur centres paramagnetic in their oxidized form (‘HiPIP-type’) was also noted. An EPR-detectable signal that may have been due to the presence of molybdenum in the electron transport chain is described and assessed. Most of the centres detected were reducible by substrate in the absence of oxidant and some were found to be wholly or partly reduced during steady state oxidation of substrates in the presence of oxygen.
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Thiosulphate Oxidation, Electron Transport and Phosphorylation in Cell-free Systems from Thiobacillus A2
More LessCell-free preparations have been made from Thiobacillus A2 that are capable of the complete oxidation of thiosulphate to sulphate and of coupling the associated electron transport to ATP synthesis with a P/O ratio of about one. Spectrophotometric measurements and the effect of electron transport chain inhibitors on cytochrome reduction and oxygen uptake indicate that thiosulphate oxidation is coupled directly to cytochrome c reduction and does not involve cytochrome b. Complete inhibition of phosphorylation by, 2,4-dinitrophenol suggests that ATP synthesis was effected exclusively by electron transport phosphorylation accompanying re-oxidation of cytochrome c. Enzymes for thiosulphate oxidation to sulphate and the reduction of cytochrome c were located in the supernatant fraction after centrifuging at 130000 g, but oxygen uptake required the 130000 g pellet fraction, which provided membrane-bound cytochrome c and cytochrome oxidase.
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Partial Purification and Resolution of a Thiosulphate-oxidizing System from Thiobacillus A2
More LessA crude thiosulphate-oxidizing extract of Thiobacillus A2 has been resolved by ammonium sulphate fractionation and DEAE-Sepharose CL-6B chromatography into fractions containing several components necessary for thiosulphate oxidation and reduction of cytochrome c. These components include two distinct c-type cytochromes, sulphite: cytochrome c oxidoreductase, rhodanese and enzymes probably responsible for thiosulphate cleavage and sulphane-sulphur oxidation. The complete cytochrome c-dependent thiosulphate-oxidizing system could be reconstituted by mixing selected fractions from the column chromatography.
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Purification and Characterization of Streptomycin 6-kinase, an Enzyme Implicated in Self-protection of a Streptomycin-producing Micro-organism
More LessStreptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5′-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mm and 0·4 mm , respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.
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Synthesis of Nitrogenase in the Cyanobacterium Gloeothece (Gloeocapsa) sp. CCAP 1430/3
More Less55FeCI3 labelling and non-denaturing gel electrophoresis were used to study nitrogenase synthesis in Gloeothece sp. CCAP 1430/3. Nitrogenase synthesis was inhibited by addition of NHX but was unaffected by elevated concentrations of O2. Upon transfer of cultures of Gloeothece from light to darkness, there was initially a slight decrease in the rate of synthesis of nitrogenase but after 4–5 h there was an almost complete cessation of synthesis. This delayed effect of darkness on nitrogenase synthesis could not be related to any change in RNA synthesis, in protein synthesis or in the rate of breakdown of storage glucan.
In cultures of Gloeothece, mRNA, including nif mRNA, was unstable, having a half-life of about 5 min. The synthesis of nif mRNA did not stop immediately upon transfer of cultures to the dark. If darkness exerts its effect on nitrogenase synthesis by inhibiting the synthesis of nif mRNA, it does so only after a lag of about 4 h.
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Stimulation of Aflatoxin Biosynthesis by Lipophilic Epoxides
More LessEpoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.
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A Chromatographic Method for the Purification of K99 Pili from Enterotoxigenic Escherichia coli
More LessDetails of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0·02 m-Tris/HCl, pH 8·6 containing 0·05 m-NaCl and DEAE-Sephadex A-50 equilibrated with 0·05 m-phosphate buffer, pH 7·2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl2 precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.
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- Biotechnology
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The Application of Materials Balancing to the Characterization of Sequential Secondary Metabolite Formation in Streptomyces cattleya NRRL 8057
More LessThe high substrate yield factor (0·73 g biomass g glucose−1) and low R.Q. (respiratory quotient, i.e. mol CO2 evolved per mol O2 consumed) value (0·8) measured during growth-phase batch cultures of Streptomyces cattleya could be rationalized in terms of the fermentation mass balance when the oxidized elemental composition of biomass was considered. R.Q. was also indicative of the sequence of secondary metabolite formation, the value rising in steps as each new product was formed. The period of maximum respiratory activity and phosphate uptake preceded maximum growth and glucose uptake. At the end of the lytic phase, a cyclopentenedione cobalt chelator was produced. The termination of lysis coincided with melanin production. Sequential cephamycin C and thienamycin production then took place. Specific hyphal protein content (per unit RNA) peaked before the production of each new metabolite. Melanin, cephamycin C and thienamycin production were initiated when glucose, ammonia and phosphate, respectively, became growth-limiting.
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- Ecology
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Ecophysiology of the Cyanobacterium Dactylococcopsis salina: Effect of Light Intensity, Sulphide and Temperature
More LessDactylococcopsis salina is a planktonic gas-vacuolated cyanobacterium that forms a distinct bacterial plate at the metalimnion of Solar Lake, Sinai. Temperature, light intensity and sulphide concentration were examined as possible limiting factors determining the distribution of D. salina during the annual limnological cycle of Solar Lake. Both laboratory cultures and in situ samples were examined for their photosynthetic activity at a wide range of temperature, light intensity and sulphide concentrations. The cyanobacterium showed a considerable light adaptation and a capacity for photosynthetic activity at high light intensities. It also showed anoxygenic photosynthesis using H2S as an alternative electron donor, but this activity was only 4% of oxygenic photosynthesis. Furthermore, H2S was highly toxic to D. salina and no CO2 photoassimilation could be detected at pH 7 and 1 mm-sulphide. Temperature is the primary environmental factor governing the distribution of D. salina in Solar Lake.
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- Genetics And Molecular Biology
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Genetic Analysis of a Gene Regulating the Timing of Developmental Events in Dictyostelium discoideum
K. Abe, Y. Okada, M. Wada and K. YanagisawaIn Dictyostelium discoideum, there exist genes which regulate the timing of developmental events. In one class of these rapidly developing (rde) mutants, the rate of development is accelerated, resulting in the formation of spores and stalk cells in about 2/3 of the time required for the parent. Linkage analysis of one of the rde strains, HTY506, demonstrated that rdeC1850, carried by the mutant is located on linkage group III. Therefore HTY506 differs genetically from the strain FR17, the first rde mutant described which carries a mutation rdeA1 on linkage group IV. While strain HTY506 has a pleiotropic phenotype, the altered characteristics of strain HTY506 appear to be caused by a single mutation, rdeC1850.
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Studies of Protoplast Fusion in Streptomyces chrysomallus
More LessConditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27–30 °C on regeneration R2 medium ( Okanishi et al., 1974 ) containing 0·25 m-sucrose. The addition of phosphate (150–300 mg 1−1) to the medium and incubation at 23 °C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.
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A Polyamine-sensitive Mutant of Aspergillus nidulans
More LessA mutation designated spsA1 has been induced in the putrescine (puA2) auxotroph of Aspergillus nidulans which enables this mutant to grow on low concentrations of spermidine in place of putrescine. In addition, the spsA1 mutant, irrespective of putrescine requirement, is abnormally sensitive to high concentrations of spermidine, spermine or the polyamine analogue methylglyoxal bis(guanylhydrazone). When spsA1 strains are grown on medium containing spermidine, uptake of the polyamine continues at a high level for a longer period than in the wild-type and leads to a doubled intracellular spermidine pool. A similar increase in the intracellular spermine pool results from growth on spermine.
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Citrate Synthase Activity in Escherichia coli Harbouring Hybrid Plasmids Containing the gltA Gene
More LessA hybrid plasmid. pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a. gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit M r was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.
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Obligatory Coupling of Colicin Release and Lysis in Mitomycin-treated Col+ Escherichia coli
More LessPrevious work has shown that Escherichia coli KH strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.
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Altered Arrangement of Proteins in the Spore Coat of a Germination Mutant of Bacillus subtilis
More LessSpores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores. The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores. However, one of the proteins (mol. wt 12000) which is normally present in the outermost layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed. The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B. subtilis chromosome, and was 47% cotransformable with leuB16. This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly. The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat.
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- Pathogenicity And Medical Microbiology
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Monoclonal Antibodies to Gonococcal Pili: Studies on Antigenic Determinants on Pili from Variants of Strain P9
More LessSeveral hybrid cell lines producing monoclonal antibodies against gonococcal pili have been derived. The reactivity of each antibody against variant pili of strain P9 was determined by ELISA. Using type-specific as well as broadly cross-reacting antibodies labelled with 125I, the capacity of unlabelled hybridoma antibodies to inhibit their attachment to various pilus types was determined. Some antibodies competed significantly and were judged to react at the same or closely positioned antigenic sites, whereas others bound non-competitively to the same pilus. These studies suggest the existence of distinct antigenic sites on gonococcal pili: a common antigenic region shared by all P9 pilus types and a type-specific region. Both antigenic sites contain more than one epitope.
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- Physiology And Growth
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Cell Cycle Initiation and Bud Emergence in Saccharomyces cerevisiae
More LessThe coordination of growth with cell division in Saccharomyces cerevisiae has been examined by investigating the order and interactions of three early events in the cell cycle. ‘Start’, which is thought to be the central control point from which alternative developmental pathways diverge ( Hartwell, 1974 ). preceded bud emergence by about 15 min regardless of cell growth rate. Cells growing in rich media showed the greatest increase in volume between ‘start’ and bud emergence. A nutrient-modulated cell size control event ( Lorincz & Carter, 1979 ) was executed coincident with, or after, ‘start’. Therefore, this latter step appears to be the primary point for cell size control in the cell cycle.
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Wall Synthesis and Assembly During Hyphal Morphogenesis in Schizophyllum commune
More LessPulse-chase experiments with [14Cglucose suggested that in growing hyphae of Schizophyllum commune, a water-soluble β-glucan is a precursor for the alkali-insoluble chitin-β-glucan complex in the wall. With microautoradiography it was found that this complex was not present at the very tip of the growing hyphae where chitin and β-glucan were inserted into the wall as individual polymers. The two polymers were then gradually turned into an insoluble complex, probably by covalent cross-linking. This was also observed in the entire hyphal apex after arrestment of growth. Evidence is presented which indicates that complex formation affects the mechanical properties of the wall, and a model is given in which the formation of a complex between chitin and β-glucan accounts for the change from plasticity towards rigidity of the wall during hyphal morphogenesis.
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Phototactic Orientation by Amoebae of Dictyostelium discoideum Slug Phototaxis Mutants
More LessWe examined the phototactic behaviour of individual amoebae of four Dictyostelium discoideum mutants which were altered in their slug phototaxis and thermotaxis. All four mutants were found to be also altered in amoebal phototaxis. This indicates that sensory processing in D. discoideum amoebae and slugs is not independent. The mutants were placed in three phenotypic classes. At suboptimal light intensities amoebal phototaxis is bidirectional, deviating right and left from the direction of incident light. Bimodality could be explained by assuming that spatial light gradients produced by internal absorption and scattering during phototaxis depend on the cell geometry in relation to the light direction.
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Growth Energetics of Rhizobium leguminosarum in Chemostat Culture
More LessThe efficiency of oxidative phosphorylation in symbiotic nitrogen-fixing bacteria of the genus Rhizobium in the root nodules of leguminous plants is of fundamental importance as it influences the efficiency of ATP generation from the oxidation of plant photosynthate for the ATP-dependent process of nitrogen fixation. A poor efficiency of energy coupling was found for Rhizobium leguminosarum grown glucose-limited in chemostat culture as indicated by low growth yields on glucose [Y max glucose = 80·3 g dry wt bacteria (mol glucose)−1 and O2 [Y max oxygen = 25·6 g dry wt bacteria (mol O2)−1. (These values are low when compared with those for aerobic heterotrophic bacteria which have two or three sites of oxidative phosphorylation.) Although H+/O measurements suggested a potential P/O ratio of 3 there was a very rapid rate of proton decay across the cell membrane; this coupled with the low growth yields suggested that the actual P/O ratio was nearer 1. Under conditions of excess dissolved oxygen cytochromes of the c, b, o and aa 3 types were detected. Under oxygen limitation a d type terminal cytochrome oxidase was also produced and was associated with a further decrease in the efficiency of energy conservation. It remains to be seen whether such a low efficiency of energy conservation is expressed by R. leguminosarum within the root nodules of a host legume.
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The Utilization of 5-Oxoproline, Ammonia and Glutamine by Rhizobium leguminosarum in Chemostat Culture
More LessThe growth in continuous culture of Rhizobium leguminosarum on the nitrogen sources glutamine, ammonia, or ammonia and 5-oxoproline (formed when aqueous glutamine solutions are autoclaved) is described. When growth was nitrogen-limited with autoclaved glutamine the specific rate of 5-oxoproline utilization was independent of dilution rate, whereas the specific rate of ammonia utilization increased with dilution rate. Although the culture was nitrogen-limited, excess nitrogen (as 5-oxoproline) was still present in the supernatant, especially at high dilution rates.
Values obtained for Y max glucose and Y max oxgen were 72·2 and 32·5 g dry wt bacteria (mol substrate)−1, respectively, under ammonia/5-oxoproline limitation and 95·7 and 30·2 g dry wt bacteria (mol substrate)−1, respectively, under glutamine limitation.
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Manganese as Substitute for Magnesium During Magnesium-limited Growth of the Cyanobacterium Anacystis nidulans
More LessAn apparent inhibition of cell division in the cyanobacterium Anacystis nidulans, caused by low Mg2+ concentrations, was abolished by increasing the medium Mn2+ concentration. Thus the mean cell volume of this organism growing in a Mg2+-limited chemostat culture decreased from an average of 1·3 to 0·4 μm3 following an increase in the reservoir Mn2+ concentration from 9·5 to 15 μm. This increase in Mn2+ had no effect on the steady-state biomass concentration, while a further elevation of the Mn2+ concentration lowered the biomass concentration, seemingly by making Mg2+ less available to the organism. The cellular Mn2+ concentration increased, while cellular Mg2+ was unaltered, following an increase in the medium Mn2+ concentration.
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Variation in Photosynthetic Membrane and Pigment Content with Light Intensity for Anacystis nidulans Grown in Continuous Cultures
More LessPhotosynthetic membrane and chlorophyll content of the cyanobacterium Anacystis nidulans were determined after growth at different light intensities in Mg2+-limited and non-Mg2+-limited continuous cultures. Decreasing the light intensity from 445 to 31 μE m−2 s−1 resulted in a threefold increase of chlorophyll per unit cell volume. The photosynthetic lamellar structure, on the other hand, increased twofold under the same conditions. The specific chlorophyll content of the photosynthetic membrane increased when the light intensity was decreased from 445 to 282 μE m−2 s−1 and was constant at lower light intensities. The results indicate that the maximum chlorophyll content in the thylakoids of A. nidulans was about 1μ5 fg m−2, which means that the membrane contained 1 chlorophyll molecule per nm2 under these conditions.
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Production and Regulation of Extracellular Endocellulase by Agaricus bisporus
K. Manning and D. A. WoodExtracellular endocellulase production by Agaricus bisporus closely paralleled mycelial growth in cultures containing microcrystalline cellulose. The enzyme was induced by various celluloses and cellobiose. In the presence of a cellulose inducer glucose and cellobiose repressed production of the enzyme. Endocellulase activity in culture filtrates was inversely related to cellulose concentration in the culture. In high concentrations of cellulose the activity of free enzyme was low. Evidence was obtained for the existence of two forms of endocellulase activity. One form adsorbed strongly to cellulose and was predominant in cultures low in cellulose. In cultures with a high cellulose content a non-adsorbable form of the enzyme was more abundant. The regulation of the appearance of free enzyme in relation to cellulose content and enzyme adsorption is discussed.
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The Relationship between Conidial Germination and Esterase Activities in Neurospora crassa
More LessEsterase activity in rapidly germinating Neurospora conidia was several times higher than the esterase activity in conidia which germinate slowly. Starch gel electrophoresis experiments demonstrated the existence of esterase isoenzymes which are specific to the conidia. These isoenzymes completely disappeared during 20 h of conidial germination at 30 °C. Electron microscopy showed the successive breakdown of electron-dense compounds in storage bodies during conidial germination. These observations, taken together, indicate that the electron-dense compounds may be hydrolysed by specific esterases to serve as an endogenous energy and material source for germ tube formation. The levels of esterase activity, however, were not always proportional to the time required for conidial germination, indicating the possibility that additional enzyme systems might also be involved in the initial stages of germination.
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A Model for Fungal Colony Growth Applied to Sclerotium rolfsii
More LessA model which relates branching kinetics to colony shape in filamentous fungi was used to study the growth of Sclerotium rolfsii. Model variables include the densities of hyphae and tips along the radius of the colony. Experiments designed to measure these variables revealed peaked distributions of hyphae, and tip densities which were concentrated just within the margin of the colony. The phenomena of lateral branching, hyphal autolysis, and anastomosis, which are known to occur in S. rolfsii, are incorporated into the model. Predictions generated by the model agree with experimental observations on this species.
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Ammonium Ion Repression of Sporulation in Saccharomyces cerevisiae
More LessThe role of NH+ 4 in the regulation of sporulation in Saccharomyces cerevisiae has been studied by analysing the effects of NH+ 4 and methylammonium ions on sporulation in a wild-type strain; a strain homozygous for the spd1 mutation, (derepressed sporulation on a number of carbon sources, reduced sensitivity to NH+ 4-inhibition of sporulation); and a strain homozygous for the gdhA6 mutation, (the structural gene for the anabolic NADP+-dependent glutamate dehydrogenase). The addition of ammonium or methylammonium ions to sporulation medium resulted in incomplete ascus formation. The gdhA6 mutation resulted in a loss of sensitivity to NH+ 4-inhibition of initiation of sporulation. At higher concentrations of NH+ 4 the strain homozygous for the gdhA6 mutation was even less sensitive than the sporulation-derepressed strain. However, in the formation of complete asci all three strains behaved very similarly over the whole range of ammonium sulphate concentration. These studies indicate there are at least two separate stages affected by NH+ 4; one early, possibly initiation, the other later, concerned with the organization and delimitation of mature spores. From the reduced sensitivity to NH+ 4-inhibition of initiation of sporulation conferred by the gdhA6 mutation, and the fact that similar results were obtained using methylammonium ion, it is concluded that some metabolite of NH+ 4 is responsible rather than NH+ 4 itself. In addition, the data provide some insight into the nature of the spdl mutation.
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Synthesis of Individual Proteins, Including Tubulins and Chloroplast Membrane Proteins, in Synchronous Cultures of the Eukaryote Chlamydomonas reinhardtii. Elimination of Periodic Changes in Protein Synthesis and Enzyme Activity under Constant Environmental Conditions
More LessChlamydomonas reinhardtii when growing under the conventional synchronizing conditions of batch culture and intermittent illumination shows periodic changes in the rate of 35SO4 uptake and in the specific rate of 35S incorporation into protein relative to total protein. There are also periodic changes in the rates of synthesis of individual proteins including several chloroplast membrane proteins and of tubulin, which is synthesized maximally prior to the initiation of division events and is not synthesized during cytokinesis. Activities of citrate synthase and aspartate transcarbamoylase enzymes increase discontinuously under synchronizing conditions. These periodic events are not part of a developmental programme which is essential for division, since the periodicity disappears under constant environmental conditions. Synchronously dividing cells then show smoothly increasing rates of 35SO4 uptake into cells and of incorporation into protein. There is a constant specific rate of isotope incorporation, relative to total protein, which correlates with the exponential accumulation of protein through the cell cycle. The 300 most abundantly labelled proteins are synthesized continuously through the cell cycle and this accords with the continuous accumulation of citrate synthase and aspartate transcarbamoylase enzyme activities. Tubulin synthesis is continuous under constant growth conditions and therefore periodic increases in tubulin concentration are not an essential part of mitotic spindle or phycoplast formation. Of the sedimentable proteins only one of the 200 most abundantly labelled shows a cessation of synthesis. A previously reported periodic synthesis of chloroplast membrane proteins is not essential for chloroplast assembly in dividing cells. The significance of these data is discussed in relation to cell cycle control and it is proposed that three categories of periodic event should be recognized. Periodic syntheses of proteins that are directly involved in division processes may be classed as primary cell cycle events if they accompany the cell cycle under all growth conditions, and these events may include division-initiating processes. If periodic synthesis of such proteins occurs only under some growth conditions then their synthesis is classed as a secondary cell cycle event. Discontinuous synthesis of proteins that do not contribute directly to division processes and are only synthesized discontinuously during or in recovery from changing environmental conditions, is considered a tertiary cell cycle event, but by adapting cell structure and metabolism to environmental conditions such events may indirectly sustain division processes. It is suggested that the periodic synthesis of tubulin under synchronizing conditions is consistent with the operation of a secondary cell cycle control, but that the periodic increases in the activity of a respiratory and biosynthetic enzyme and the periodic synthesis of numerous individual unidentified proteins, under changing conditions in synchronous culture are only tertiarily related to the cell cycle.
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- Taxonomy
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Numerical Classification of Streptomyces and Related Genera
More LessFour hundred and seventy-five strains, which included 394 type cultures of Streptomyces and representatives of 14 other actinomycete genera, were studied. Overall similarities of these strains for 139 unit characters were determined by the S SM and S J coefficients and clustering by the UPGMA algorithm. Test error and overlap between the phena defined were within acceptable limits. Cluster-groups were defined by the S SM coefficient at the 70·1% similarity (S) level and by the S J coefficient at the 50% S-level. Clusters were distinguished at the 77·5% S SM and 63% S J S-levels. Groupings obtained with the two coefficients were generally similar, but there were some changes in the definition and membership of cluster-groups and clusters.
The phenetic data obtained, together with those from previous diverse studies, indicated that the genera Actinopycnidium, Actinosporangium, Chainia, Elytrosporangium, Kitasatoa and Micro-ellobosporia should be reduced to synonyms of Streptomyces, while Intrasporangium, Nocardioides and Streptoverticillium remained as distinct genera in the family Streptomycetaceae. Nocardiopsis dassonvillei also showed strong phenetic affinity to Streptomyces, despite its chemotaxonomic differences. Actinomadura sensu stricto was phenetically distinguishable from Streptomyces and ‘Nocardid’ mediterranea was recognized as a taxon distinct from both these genera and from Nocardia sensu stricto.
Most of the Streptomyces type cultures fell into one large cluster-group. At the 77·5% S SM S-level, they were recovered in 19 major and 40 minor clusters, with 18 strains recovered as single member clusters. The status of the latter as species was therefore confirmed. Most of the minor clusters, consisting of two to five strains, can also be regarded as species. The major clusters varied in size (from 6 to 71 strains) and in their homogeneity. Therefore, it is suggested that they be regarded as species-groups until further information is available. The results provide a basis for the reduction of the large number of Streptomyces species which have been described. They also demonstrate that the previous use of a limited number of subjectively chosen characters to define species-groups or species has resulted in artificial classifications.
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A Probability Matrix for Identification of some Streptomycetes
The character state data obtained for clusters defined at the 77·5% S SM similarity level in the phenetic numerical classification described by Williams et al. (1983) were used to construct a probabilistic identification matrix. The 23 phena included were the major clusters (19 Streptomyces, 2 Streptoverticillium and ‘Nocardia’ mediterranea) and one minor cluster (Streptomyces fradiae). The characters most diagnostic for these clusters were selected using Sneath’s CHARSEP and DIACHAR programs. The resulting matrix consisted of 41 characters × 23 phena.
Identification scores, determined by Sneath’s MATIDEN program were used to evaluate the matrix. Theoretical assessment was achieved by determination of the cluster overlap (OVERMAT), the identification scores for the Hypothetical Medium Organism of each cluster (MOSTTYP), and the scores for randomly selected cluster representatives using the classification data of Williams et al. (1983) . The matrix was evaluated practically by the independent re-determination of the characters for the same cluster representatives, which-also provided a measure of test error. Finally it was used to identify unknown isolates from a range of habitats.
The results showed that the matrix was theoretically sound. Test error was within acceptable limits and did not distort identifications. Of the unknown isolates, 80% were clearly identified with a cluster. It is suggested that the matrix could form the basis for a more objective identification and grouping of the large number of Streptomyces species which have been described.
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A Phylogenetic Analysis of the Family Dermatophilaceae
More LessThe comparative analysis of the 16S ribosomal ribonucleic acid (rRNA) of Geodermatophilus obscurus DSM 43060 and Dermatophilus congolensis DSM 43037 revealed that these members of the family Dermatophilaceae were only remotely related. While G. obscurus represented an individual and separate line of descent within the phylogenetically defined order Actinomycetales, D. congolensis was closely related to representatives of Arthrobacter, Micrococcus, Cellulomonas, Brevibacterium, Promicromonospora and Microbacterium.
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Intraspecies Genetic Relatedness among Strains of Acholeplasma laidlawii and of Acholeplasma axanthum by Nucleic Acid Hybridization
More LessThis study compares the intraspecies genetic relatedness among strains of two established species of Acholeplasma. Radiolabeled DNA probes were prepared from three strains of Acholeplasma laidlawii and two strains of Acholeplasma axanthum, by using the nick translation method. The labelled DNA probes of these two strains were hybridized to an excess of unlabelled DNA from 12 strains of Acholeplasma laidlawii and from six strains of Acholeplasma axanthum, respectively. Nucleic acid hybridization analyses showed a wide variation among strains within each of the two established species, ranging from 48 to 100% homology. The results demonstrate that strains isolated from diverse hosts and habitats within a given species of Acholeplasma exhibit extensive genotypic variations.
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DNA Cleavage Patterns as Indicators of Genotypic Heterogeneity among Strains of Acholeplasma and Mycoplasma Species
More LessElectrophoretic patterns of digestion products of Acholeplasma and Mycoplasma DNA by restriction endonucleases were compared. The patterns of Acholeplasma axanthum strains isolated from a variety of hosts and habitats differed markedly from each other, indicating considerable genotypic heterogeneity among strains included in this species. Heterogeneity was less marked among the Acholeplasma oculi strains tested, and was minimal among strains of the avian pathogen Mycoplasma gallisepticum. Strains of Mycoplasma genitalium isolated from the urethra of patients with non-gonococcal urethritis and from the urethra of an experimentally infected chimpanzee yielded identical cleavage patterns, indicating a high degree of genetic homogeneity of these strains. The data support the notion that mycoplasma species of strict host and tissue specificity exhibit marked genetic homogeneity. The advantages and deficiencies of the use of DNA cleavage patterns for classification purposes are discussed.
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Mycoplasma-like Organisms from Plants with ‘Yellows’ Diseases Lack a Spiroplasma-specific Antigen
More LessAn antigen which is specific to Spiroplasma and was unaffected by changes in cell morphology was used to demonstrate the presence of spiroplasmas in diseased plant material. The same antigen could not be detected in plants infected with eight different ‘yellows’ diseases with which non-cultivable mycoplasma-like organisms were associated.
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