- Volume 130, Issue 8, 1984
Volume 130, Issue 8, 1984
- Biochemistry
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An Analysis of Intracellular Proteolytic Activities of Tetrahymena pyriformis GL
More LessFive different assay conditions were used to analyse the intracellular proteolytic system of Tetrahymena pyriformis GL. Acid proteinase activity was measured using hide powder azure at pH4 and neutral proteinase activity was measured using hide powder azure and azocasein at pH 8 and two peptide nitroanilides at pH 7. Differences between the activities were apparent from their sensitivity to proteinase inhibitors although all were likely to be due to cysteine proteinases. Indeed all were enhanced by the presence of 1 mm-DTT. The neutral activities could be resolved into at least three forms by ion-exchange chromatography, each form having a different specificity for the substrates used. The acid proteinase activity was similarly separated into different fractions. The intracellular activity of acid proteinase responded differently from that of the neutral activities to culture age or resuspension of cells in fresh medium and starvation buffer. Furthermore, analysis on sucrose density gradients showed that although the neutral activities had a similar subcellular distribution to one another this differed from that of the acid proteinase activity. This suggests that the physiological roles of the acid and neutral proteinases may differ. PAGE using gels containing haemoglobin to detect proteinases was less successful than with other species of protozoa. However, it did reveal multiple forms of acid proteinase.
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Studies on Heterogeneous Lipopolysaccharide Fractions of Vibrio cholerae 569B
More LessBy SDS-PAGE or gel filtration on Sephadex G-25, lipopolysaccharide (LPS) isolated from Vibrio cholerae 569B (Inaba) can be separated into two distinct fractions, one corresponding to smooth LPS and the other to rough LPS. Pulse-labelling of LPS with [14C]glucose showed that the rough form is synthesized first followed by the biosynthesis of the smooth form. A preferential release of the smooth LPS of V. cholerae 569B was also detected during normal growth of cells.
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Chloroperoxidases from Caldariomyces (= Leptoxyphium) Cultures: Glycoproteins with Variable Carbohydrate Content and Isoenzymic Forms
More LessTwenty-six fungal cultures, including ten Caldariomyces (= Leptoxyphium) cultures and five other capnodiaceous fungi, were examined for extracellular chloroperoxidase production on four growth media. Only the Caldariomyces cultures produced enzyme activity and enzyme production was highest on growth in a phytone medium. Chloroperoxidase was partly purified from the ten Caldariomyces cultures. All preparations were glycoproteins, with different carbohydrate content. The absorption spectra of the ten samples were indistinguishable and this was reflected in similar Rz (A 403/A 280) values. SDS-PAGE revealed two major bands of protein in all preparations but in different proportions: staining for carbohydrate confirmed these bands to be glycoproteins. PAGE at pH 3 showed each preparation to be composed of several isoenzymes and these could be grouped according to their migration patterns. Kinetic constants, where determined, and pH optima showed no differences among the ten preparations and all exhibited catalase and peroxidase activities in the same relative proportions to chlorinating activity.
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Subcellular Segregation of Phosphoribulokinase and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in the Cyanobacterium Chlorogloeopsis fritschii
More LessThe Calvin cycle enzyme phosphoribulokinase has been localized in terms of catalytic activity and enzyme protein in the cyanobacterium Chlorogloeopsis fritschii. In contrast to the CO2-fixing enzyme of the Calvin cycle, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which occurs in the cytoplasm and in the carboxysomes, phosphoribulokinase is essentially (95%) cytoplasmic in extracts from cells grown photoautotrophically to late exponential phase. Total phosphoribulokinase accounted for 0·6% of total cell protein in these cells. Immunochemical identity was found between the phosphoribulokinase located in the cytoplasm and the remaining enzyme (5%) associated with the particulate fraction of the cell obtained following differential centrifugation. On density gradient centrifugation of the particulate fraction into Percoll plus sucrose the RuBisCO activity was located in a carboxysome band in the lower half of the gradient while the phosphoribulokinase activity essentially remained concentrated at the top of the gradient. Comparison of RuBisCO and phosphoribulokinase distributions suggests that the latter is not a carboxysomal enzyme in late exponential phase cells of C. fritschii.
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- Development And Structure
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Lysis of Vibrio cholerae Cells: Direct Isolation of the Outer Membrane from Whole Cells by Treatment with Urea
More LessCells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.
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Modes of Cell Division and Branch Formation in the Morphogenesis of the Cyanobacterium Mastigocladus laminosus
More LessPatterns of morphogenesis in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined by light and electron microscopy. Morphologically different types of filaments possessed, and were restricted to, distinct modes of cell division. Division in narrow and lateral branch filaments was always perpendicular to the long axis of the filament, indicating that these filaments were involved principally with trichome elongation. Division in wide filaments was either diagonal or (more often) parallel to the long axis of the filament, the latter enabling production of lateral branches. Cells of filaments in transition between the narrow and wide forms changed in shape and size, but divided only rarely. Wide cells that produced lateral branches were ultrastructurally identical to the other cells in wide filaments and were not surrounded by sheath material; branches did not arise by germination of akinetes. The morphological and ultrastructural characteristics of successive branch-filament cells (starting proximal to the parental filament) ranged from those typical of wide-filament cells to those typical of narrow-filament cells. Wide cells differed ultrastructurally from narrow cells in containing fewer ribosomes, less nuclear material, and more reserve materials. Wide cells lacked characteristic ultrastructural features of akinetes or other resting cells, appearing instead to be active vegetative forms. Lateral branches were released from parental filaments by deterioration of specific wide-filament cells.
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- Genetics And Molecular Biology
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Induction of Bacteriophage from Members of the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum Serocomplex
More LessBacteriophages have been induced from strains in the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum serocomplex by exposure of cultures to UV light or treatment with mitomycin C. One-sixth of the strains examined, representing all but one of the 31 authenticated serotypes, were found to possess phages lytic for a Mycobacterium smegmatis indicator strain. Four single-plaque isolated phages, TM4, TM9, TM10 and TM20, were purified and shown to have a similar morphology on electron micrographs. They had an isometric head of diameter 50–58 nm and a flexible non-contractile tail about 170 nm in length with a terminal bulb. All had an identical buoyant density in CsCl of 1·521 g cm−3 and extreme sensitivity to chloroform. The induced phages differed in host range and possessed the unique ability to lyse other members of the serocomplex. Interest in these phages centres on a possible role in mediating genetic interrelationships between members of the serocomplex.
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Isolation of a DNA Plasmid in the Fungus Rhizoctonia solani
More LessA DNA plasmid, designated pRS64, was detected in three isolates of anastomosis group 4 (AG-4) of Rhizoctonia solani Kühn by biophysical methods. The plasmid was a linear double-stranded DNA with a molecular weight of 1·68 ± 0·06 × 106 or 2617 ± 87 bp. Weakly pathogenic isolates of R. solani, 1668 RI-1, 1271 RI-64 and 1272 RI-1, which showed abnormally slow growth, contained the plasmids, but pathogenic isolates, 1668, 1271 and 1272, showing normal growth, contained no detectable plasmid DNA.
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Linkage Analysis of Two Phagocytosis Receptor Loci in Dictyostelium discoideum
More LessTwo recessive mutations of the cellular slime mould Dictyostelium discoideum, phgA1356 and phgB1351, which express altered phagocytic properties, were characterized using parasexual genetic analysis. The phgA locus was assigned to linkage group VII, and the phgB locus to linkage group IV. Complementation analysis of a further twelve independently derived phagocytosis mutants showed that each fell into one of these two complementation groups.
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Genetic Analysis of a Dictyostelium discoideum Mutant Resistant to Adenosine 3′:5′-Cyclic Phosphorothioate, an Inhibitor of Wild-type Development
More LessA mutant of Dictyostelium discoideum strain AX2 with altered responses to cyclic AMP has been analysed genetically. The mutant, HG302, aggregates and fruits in the presence of adenosine 3′:5′-cyclic phosphorothioate (cAMPS). This slowly hydrolysed analogue of cyclic AMP inhibits wild-type development by blocking cell differentiation from the growth phase to the aggregation-competent stage. The mutant forms small aggregates in the absence and in the presence of cAMPS, and is further distinguished from the wild type by decreased activities of extracellular phosphodiesterase and phosphodiesterase inhibitor. These phenotypic characteristics were all linked to the cAMPS resistance, which was assigned to linkage group II. Mitotic crossover experiments showed that the cAMPS-resistance mutation, casA1000, maps proximal to the acrA locus in the neighbourhood of whiA. The data suggest that casA1000 acts pleiotropically, and that the product of the wild-type allele is required in a part of the signal-processing pathway common to control of development and regulation of extracellular phosphodiesterase.
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Genetic and Phenotypic Characterization of a Cluster of Mutations in the spoVA locus of Bacillus subtilis
More LessTwenty-nine mutants blocked during stage V of sporulation have been isolated following directed mutagenesis of the lys-1 region of the Bacillus subtilis 168 chromosome. All of a sample of eight mutants tested are unaffected in sporulation marker events up to stage IV but did not produce dipicolinic acid. They produced stable ‘phase white’ spores that were released from the mother cell, and were partially resistant to toluene and lysozyme but sensitive to chloroform and heat. Mutation spoVA89, known to be in the lys-1 region, showed similar phenotypic characteristics. Three-factor transformation crosses and recombination indices showed that the new mutations and spoVA89 lie in a single linkage group, which maps between lys-1 and another sporulation locus, spoIIA. The size of the spoV A locus is such that it probably contains several genes, and these may be contiguous with the cluster of genes included within the spoIIA locus.
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Use of Integrational Plasmid Vectors to Demonstrate the Polycistronic Nature of a Transcriptional Unit (spoIIA) Required for Sporulation of Bacillus subtilis
More LessPlasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B. subtilis strain, BR 151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome. Two such plasmids, pPP35 and pPP36, yielded Spo-transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit. The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic. This is the first demonstration of such a polycistronic sporulation transcriptional unit. The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames. Two other plasmids yielded Spo+ transformants of BR 151, and these define the outer limits of the transcriptional unit. The adjacent sporulation locus identified by the spoVA89 mutation was not part of the same transcriptional unit.
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Cloning of the Bacillus subtilis spoIIA and spoVA Loci in Phage ϕ105DI:1t
More LessA 6·95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage ϕ105DI:lt. The recombinant phage (ϕ105DS1) contained DNA of 33·8 kb as compared with 35·2 kb for ϕ105DI:lt and 39·2 kb for the wild-type phage. In the presence of helper phage, ϕ105DS1 complemented both spoIIA and spoVA mutations in B. subtilis.
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Nucleotide Sequence of Sporulation Locus spoIIA in Bacillus subtilis
P. Fort and P. J. PiggotWe have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.
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Construction and Characterization of Recombinant Phage 𝜙105 d(Cmr met) for Cloning in Bacillus subtilis
More LessA 1·6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage ø105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted ø105 d(Cmr met), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.
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- Physiology And Growth
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The Use of Phenolic Glycosides for Studying the Aerobic or Anaerobic Transport of Disaccharides into Yeasts
More LessNitrophenolic glycosides have been used to characterize the kinetics of anaerobic transport into yeasts. This procedure overcomes the problem of providing energy for anaerobic transport, when using non-metabolizable sugar analogues. The glycosides were hydrolysed intracellularly, the glycon catabolized, and the nitrophenol rapidly expelled from the cells. Transport was the rate-limiting metabolic step and involved proton symport. Experiments are described that establish the validity of the method, which is simple, quick, cheap and reliable, and seems generally applicable to yeasts. The high absorption coefficients of the nitrophenols allow precise measurements to be made at low concentrations, at which the proton-conducting properties of the nitrophenols are minimal.
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Magnesium and the Regulation of Germ-tube Formation in Candida albicans
More LessCandida albicans requires Mg2+ for germ-tube formation. Mg-deficient media, metal ion chelators and the ionophore A23187 inhibited germ-tube formation. Cell Mg content during exponential yeast-phase growth remained constant but increased throughout germ-tube formation. The onset of germ-tube formation coincided with a sharp peak in Mg concentration within the cells. Yeast-phase cells of strain CA2, which did not form germ-tubes, had a lower Mg content and failed to accumulate Mg when incubated under conditions for germ-tube formation. Mg also increased the uptake and incorporation of N-acetylglucosamine. These findings point to a central regulatory role for Mg in C. albicans morphogenesis.
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Regulation of the Key Enzymes of Methylated Amine Metabolism in Candida boidinii
More LessNitrogen assimilation during growth of Candida boidinii on methylated amines as sole nitrogen source involves NADP-dependent glutamate dehydrogenase. Changes in enzyme activities during the adaptation of the yeast from growth on ammonium to growth on trimethylamine were examined. No ammonia, dimethylamine or monomethylamine could be detected in the medium during growth on trimethylamine. When two methylated amines were supplied together, they were used simultaneously, although monomethylamine was metabolized more quickly than the others. When cells were grown on a low concentration of ammonium plus higher concentrations of di- or trimethylamine, the ammonium was used first. NADP-dependent glutamate dehydrogenase was the first enzyme to be derepressed, followed by methylamine oxidase and formaldehyde dehydrogenase. Di- and trimethylamine mono-oxygenase activities only appeared when the ammonium concentration fell below 0.5 mM. At this point amine utilization could be detected and no diauxic lag was observed in the growth curve. During growth on limiting ammonium, there was an increase in the activity of methylamine oxidase (150-fold) and catalase (5-fold) in the absence of any amine, but no amine mono-oxygenase activity was detected. Addition of ammonium ions to cultures growing on dimethylamine produced an immediate repression of synthesis of methylamine oxidase, NADP-dependent glutamate dehydrogenase and the two amine mono-oxygenases. An inverse correlation was found between intracellular ammonium concentration and methylamine oxidase activity. Ammonium ions also inhibited the uptake of dimethylamine or trimethylamine by washed suspensions of dimethylamine-grown cells. It is concluded that the control of methylamine oxidase and catalase and (independently) of NADP-dependent glutamate dehydrogenase is by repression of enzyme synthesis by ammonium, while expression of amine mono-oxygenases seems to require the amine to be present in the medium. Formaldehyde and formate dehydrogenases seem also to be induced by their respective substrates.
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Methylamine and Ammonium Transport Systems in Rhizobium leguminosarum MNF3841
More LessAs the sole source of nitrogen, methylamine supported the growth of a range of species of Rhizobium. The methylamine assimilatory system was inducible in R. leguminosarum MNF3841, whereas the capacity to utilize NH4 + as a nitrogen source was constitutive. An uptake system for [14C]methylamine (methylamine permease) was induced by growth of MNF3841 on methylamine or ethylamine. The uptake was sensitive to 2,4-dinitrophenol, azide and carbonyl cyanide m-chlorophenylhydrazone. The methylamine permease had a K m of 0·035 mm, a V max of 2·2 nmol min−1 (mg protein)−1 and a K i for ammonium of 1·5 mm. Most of the [14C]methylamine accumulated by cells was rapidly incorporated into TCA-insoluble materials. An NH4 +-sensitive methylamine-accumulating system distinct from the methylamine permease was demonstrated in ammonia-limited cells grown in continuous culture. This system, the ammonium permease, had a K m of 0·11 mm (for methylamine), a K i for NH4 + of 0·007 mm and a V max, of 2·5 nmol min−1 (mg protein)−1. Methylamine was accumulated by chemostat-grown, N-limited cells and could exchange with unlabelled methylamine. Treatment with carbonyl cyanide m-chlorophenylhydrazone caused efflux of the accumulated methylamine, whereas high concentrations of NH4 + did not. Thus R. leguminosarum possesses a specific methylamine permease which is quite distinct from the ammonium permease.
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The Osmotic Responses of Penicillium ochro-chloron: Changes in Internal Solute Levels in Response to Copper and Salt Stress
More LessThe marked copper tolerance of Penicillium ochro-chloron has been confirmed as has its ability to grow in solutions of high salinity. The major low molecular weight organic solutes present in P. ochro-chloron were glycerol, erythritol, arabitol, mannitol, sorbitol and trehalose. Of these, glycerol increased significantly during growth in high concentrations of Na+ or Cu2+, a 15-fold increase, relative to the control, occurring in concentrations of 0.5 M. Cell K+ increased with elevated external Na+ but decreased slightly with elevated external Cu2+. With high external Cu2+ mycelium maintained a constant level of Cu2+, with low levels of Na+ in all treatments. It is concluded that the high concentrations of glycerol induced by high external Na+ or Cu2+ had a significant osmotic effect, allowing growth in media of high osmolality. The exclusion of ions may have a gratuitous role in the copper tolerance of P. ochro-chloron.
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Mixotrophic Capabilities of Alcaligenes eutrophus
More LessThe facultatively chemolithoautotrophic hydrogen bacterium Alcaligenes eutrophus was able to utilize organic and inorganic substrates concomitantly, i.e. to grow mixotrophically. The mixotrophic capabilities were investigated under succinate-limited growth of A. eutrophus with molecular hydrogen in a gas atmosphere devoid of carbon dioxide. At a dilution rate (D) of 00·2 h−1, the mixotrophic cellular yield was increased by 135% over the heterotrophic yield with succinate alone. Total carbon analysis revealed that under these conditions 95% of the succinate carbon was converted to cell carbon. The mixotrophic yield decreased only slightly at dilution rates lower than 00·2 h−1 but significantly at higher dilution rates and was only 18% above the heterotrophic yield at D = 00·32 h−1. Unlike other facultative chemoautotrophs, mixotrophic growth of A. eutrophus required both H2 oxidation (Hox) and autotrophic CO2 fixation (Cfx), as evident from mutants defective in either H2 oxidation (Hox−) or autotrophic metabolism (Cfx−), as well as from incorporation studies of radioactive substrates. The cellular yield of a Cfx− mutant, HF17, increased only slightly (by 14%) upon the addition of H2, indicating that the ability of A. eutrophus to change the metabolism of a heterotrophic substrate was limited. Hox− mutants did not increase their cellular yield under identical growth conditions.
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Photosynthesis and Inorganic Carbon Uptake by Spheroplasts Isolated from the Cyanobacterium Anabaena variabilis
More LessSpheroplasts were prepared from the filamentous cyanobacterium Anabaena variabilis by lysozyme treatment followed by mild sonication. The rate of photosynthesis and the initial rate of accumulation of inorganic carbon within the spheroplasts were very close to those observed in intact cells. The steady-state intracellular inorganic carbon pool, however, was smaller in spheroplasts than in intact cells. It is suggested that the cell wall does not play an essential role in the process of HCO3 − transport (leading to accumulation of inorganic carbon internally) but may have a significant impact on the diffusional dissipation of the intracellular pool of inorganic carbon.
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The Antibacterial Action of Tinopal AN
More LessThe bactericidal activity of Tinopal AN [1,1-bis(3,N-5-dimethyl-benzoxazol-2-yl)-methine p-toluene sulphonate] was shown to be due to a mechanism entirely independent of its inhibitory effects upon NADH dehydrogenase which were reported previously. Whereas the compound had no significant effect upon DNA synthesis in Escherichia coli D22, RNA and protein synthesis were immediately and markedly inhibited. In confirmation, Tinopal AN caused an immediate cessation in inducible β-galactosidase synthesis in the same organism. An in vitro assay of the transcription of calf-thymus DNA by purified E. coli RNA polymerase showed that this process was inhibited by Tinopal AN.
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Restoration of Aerial Mycelium and Antibiotic Production in a Streptomyces griseoflavus Arginine Auxotroph
More LessAn arginine auxotrophic mutant was obtained from Streptomyces griseoflavus (bicozamycin-producing strain). The mutant grew on synthetic agar supplemented with either arginine, ornithine, citrulline or argininosuccinate, but produced massive aerial mycelium and bicozamycin only with citrulline. In liquid culture, citrulline also completely restored the ability of the mutant to produce bicozamycin. Culture with arginine or ornithine markedly changed intracellular pools of these ornithine-cycle amino acids, but did not affect the other amino acid pools. The ability to produce antibiotic (but not that to form aerial mycelium) was partially restored by certain mutations to ethionine resistance (Eth-1 and Eth-2). These mutations caused decreased or increased S-adenosylmethionine synthetase activity, but both resulted in a 4·5–8-fold increase in the intracellular S-adenosylmethionine pool. Exogenous addition of S-adenosylmethionine (0·5–3 mM) also partially restored the antibiotic-producing ability of the arginine auxotroph. No difference in the S-adenosylmethionine pool was observed in organisms grown with arginine and citrulline. It was suggested that citrulline and S-adenosylmethionine are somehow involved in the initiation of differentiation and secondary metabolism of S. griseoflavus.
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Effect of Growth Rate on Streptomycin Accumulation by Escherichia coli and Bacillus megaterium
More LessThe rate of accumulation of streptomycin by streptomycin-sensitive strains of Escherichia coli and Bacillus megaterium, grown in chemostats, was related to the growth rate prior to addition of the antibiotic. For E. coli the length of the lag period that preceded accumulation was also growth rate-dependent. Thus faster growing cultures accumulated streptomycin more rapidly and with a shorter lag than slower growing cultures. The rate of efflux of streptomycin from bacteria that had accumulated streptomycin was not greatly influenced by growth rates of the cultures. At a particular growth rate, accumulation of streptomycin was found to be faster at higher concentrations of the antibiotic. Rapid accumulation of streptomycin was not observed with continuous cultures of a streptomycin-resistant strain of E. coli. Accumulation of streptomycin was abolished when growth was inhibited by either terminating the flow of fresh medium to a chemostat or by adding inhibitors that block protein synthesis. These results suggest that the rate of accumulation of streptomycin is related to the concentration of streptomycin-sensitive ribosomes that are actively engaged in protein synthesis within the bacterial cells.
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Microthrix parvicella, a Filamentous Bacterium from Activated Sludge: Growth on Tween 80 as Carbon and Energy Source
More LessMicrothrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0·32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1·3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0·016 h−1 but was affected by the concentration of Casamino acids in the medium.
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Regulation of Carotenogenesis by Inorganic Phosphate in Blakeslea trispora
More LessIncreasing the inorganic phosphate concentration of the medium from 0·01% to 1% (w/v) resulted in 4·5-fold higher production of carotene in Blakeslea trispora. The pattern of β-carotene synthesis in B. trispora remained essentially the same in both high (1%) and low (0·01%) phosphate grown cultures. Higher concentrations of intracellular orthophosphate and lower activities of acid and alkaline phosphatase were found in the high phosphate grown cells as compared to the low phosphate grown cultures. The intracellular acid and alkaline phosphatase activities of B. trispora were competitively inhibited by inorganic phosphate. One or more forms of the acid and the alkaline phosphatases were apparently repressed during active carotenogenesis in the high phosphate grown B. trispora as compared to low phosphate grown cells. An inverse relationship was found between carotenogenesis and the intracellular phosphatase activities and it is suggested that inorganic phosphate concentration affects β-carotene synthesis in B. trispora by regulating the intracellular phosphatase levels in the culture.
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The Co-ordination of Chitosan and Chitin Synthesis in Mucor rouxii
More LessChitin synthetase preparations from cell walls and chitosomes of the fungus Mucor rouxii were tested for their ability to synthesize chitosan when incubated with uridine diphosphate N-acetyl-D-glucosamine in the presence of chitin deacetylase. The most effective chitin synthetase preparation was one dissociated from cell walls with digitonin. The rate of chitosan synthesis by the wall-dissociated chitin synthetase was about three times that of an equivalent amount of cell walls. The chitosan-synthesizing ability of chitosomes was relatively low, but was more than tripled by treatment with digitonin. Presumably, digitonin improves chitosan yields by dissociating chitin synthetase. The dissociated enzyme would produce dispersed chitin chains that could be attacked by chitin deacetylase before they have had time to crystallize into microfibrils. The regulation of chitin and chitosan syntheses in vivo may be determined by the organization of chitin synthetase molecules at the cell surface. Those molecules that remain organized as a complex, similar if not identical to that found in chitosomes, would produce mainly chitin. Chitosan would be preferentially produced by chitin synthetase molecules which are dispersed upon reaching the cell surface.
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- Systematics
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Mycobactins as Chemotaxonomic Characters for Some Rapidly Growing Mycobacteria
More LessThirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, M. diernhoferi, M. duvalii, M. flavescens, M. fortuitum, M. gadium, M. gallinarum, M. neoaurum, M. parafortuitum, M. peregrinum, M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and M. kanazawa failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and M. peregrinum) formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, M. diernhoferi, M. vaccae and M. parafortuitum). The mycobactin from M. gallinarum was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.
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Numerical Taxonomy of Erwinia Species Using API Systems
More LessAPI 20E, API ZYM and eight other enzymic API systems were tested on 123 strains belonging to 18 Erwinia species, six Enterobacter agglomerans strains and 22 reference strains belonging to other phytopathogenic genera and other enterobacterial species. The data obtained, from a total of 130 tests, were subjected to numerical analysis. Test reproducibility within the API 20E system varied from 88 to 100%. The numerical analysis revealed 12 phenons; in six of these phenons two or three subphenons could be differentiated. Several of these (sub)phenons corresponded to established Erwinia species and could be differentiated from each other by 25 characters. No clearcut distinction could be made between the ‘amylovora’, ‘carotovora’ and ‘herbicola’ groups. Seven phenons were further analysed with the API 50CHE system. The results provided evidence for the retention of Er. quercina, Er. nigrifluens, Er. salicis, Er. amylovora, Er. rubrifaciens, Er. mallotivora, Er. stewartii, Er. cypripedii and Er. chrysanthemi as separate taxa and supported the synonymy within the pairs Er. ananas and Er. uredovora, Er. dissolvens and Enterobacter cloacae, Er. carotovora subsp. atroseptica and Er. carotovora subsp. carotovora, Er. milletiae and one of the Er. herbicola clusters. The inadequacy of the present classification of several Erwinia species, such as Er. herbicola and Er. rhapontici, is highlighted. The results show that API systems are a useful and rapid alternative to conventional phenotypical testing for the classification and identification of Erwinia species.
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The Ribonucleotide Sequence of 5S rRNA from Two Strains of Deep-Sea Barophilic Bacteria
More LessDeep-sea bacteria were isolated from the digestive tract of animals inhabiting depths of 5900 m in the Puerto Rico Trench and 4300 m near the Walvis Ridge. Growth of two bacterial strains was measured in marine broth and in solid media under a range of pressures and temperatures. Both strains were barophilic at 2 °C (± 1 °C) with an optimal growth rate of 0·22 h-1 at a pressure 30% lower than that encountered in situ. At 1 atm they grew at temperatures ranging from 10·2 to 180·2 °C (± 0·3 °C), while in situ pressures increased the upper temperature limit to 230·3 °C. Both strains were identified as members of the genus Vibrio, based on standard taxonomic tests and mol % G + C values (470·0 and 470·1). Ribonucleotide sequences determined for 5S ribosomal RNA from each strain confirmed relationship to the Vibrio-Photobacterium group, as represented by V. harveyi and P. phosphoreum, but the barophiles were clearly distinct from these species. Secondary structure conformed to the established model for eubacterial 5S rRNA.
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A Numerical Classification of Fission Yeasts of the Genus Schizosaccharomyces Lindner
More LessA numerical classification of the yeast genus Schizosaccharomyces was undertaken using 60 strains and 100 characters. Three distinct clusters were observed, corresponding to S. pombe (SSM = 83%), both varieties of S. japonicus (SSM = 78%), and S. octosporus(SSM = 75%). Schizosaccharomyces malidevorans and S. slooffiae, each of which is available only as the type, could not be differentiated from S. pombe and S. octosporusrespectively.
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Restriction Enzyme Analysis of Bacillus subtilis Bacteriophage 𝜙105 DNA
More LessThe recognition sites on 𝜙105 DNA for the restriction endonucleases EcoRI, BglII, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from 𝜙105 contains no recognition sites for the endonucleases BamHI and XbaI.
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