- Volume 128, Issue 11, 1982
Volume 128, Issue 11, 1982
- Genetics And Molecular Biology
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Phages Iα and I2-2: IncI Plasmid-dependent Bacteriophages
More LessPhage Iα was isolated from sewage from Windhoek, South West Africa. It formed relatively clear plaques about 2 mm in diameter, on sensitive strains of Escherichia coli K12 and Salmonella typhimurium LT2. The phage had an hexagonal outline with a diameter of about 24 nm, contained RNA and was resistant to chloroform. Phage Iα formed plaques or propagated only on organisms carrying I1 plasmids or the Iγ plasmid R621a. The efficiency of plating was higher on E. coli than on S. typhimurium hosts. The phage adsorbed along the length of shafts of I1 pili.
Phage I2-2 was isolated from Pretoria sewage. It was a filamentous virus and individual virions varied considerably in length. Phage I2-2 formed turbid plaques which varied from pin point to about 1 mm in diameter on all hosts. It was resistant to RNAase and sensitive to chloroform. Phage I2-2 had a spectrum of activity limited to strains harbouring I2 plasmids but the adsorption site could not be demonstrated. The phage was not related serologically to phages If1 or PR64FS.
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Isolation and Characterization of a Recombinant Plasmid Carrying a Functional Part of the Bacillus subtilis spoIIA Locus
More LessPlasmid pHM2 consists of a 3·3 kb insert of Bacillus subtilis DNA in the chimeric plasmid pHV33, and can replicate in Escherichia coli and B. subtilis. In B. subtilis, pHM2 complements the defects resulting from mutations spo-42, spo-50, spo-69 and sas-1 in the spoIIA locus. This complementation can occur in recE4 strains where recombination of the plasmid with the chromosome is prevented, and the chromosome retains the mutant allele. Thus the plasmid carries a functional part of the spoIIA locus; it does not contain the complete locus as it cannot complement several other spoIIA mutations. It is likely that the locus is complex, containing at least two genes.
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- Pathogenicity And Medical Microbiology
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Carbohydrate Mediation of the Biological Activities of the Glycolipoprotein of Pseudomonas aeruginosa
More LessThe glycolipoprotein (GLP) extracted from the surface slime of Pseudomonas aeruginosa produces effects in mice similar to those of the viable cell. The lethal activity has been located in the lipid moiety; however, degradation of the carbohydrate moiety with sodium metaperiodate reduced the antigenicity and abolished the lethality of the GLP. Similar degradation with a phage-induced polysaccharide depolymerase reduced the antigenicity only slightly but reduced the lethality over 60%. The neutral sugar composition of the isolated polysaccharide moiety was shown to be that of the parental GLP. Of the component neutral sugars, mannose and its derivatives were capable of inhibiting the agglutination of erythrocytes coated with GLP. Inhibition also occurred with a soluble mannose polymer from the cell walls of yeast. Antiserum to GLP and to its isolated polysaccharide moiety agglutinated yeast cells, whereas antiserum to a glycolipid fragment of the GLP lacking mannose did not. The lethality of the GLP was reduced by degradation with α-mannosidase or by blocking the mannose residues with concanavalin A, and the glycolipid fragment showed less lethality than the native GLP. We conclude that mannose, in addition to being an immunodominant sugar, is an effector sugar in the expression of GLP lethality.
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- Physiology And Growth
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On the Growth and Form of Escherichia coli
More LessThe shape of many micro-organisms can be understood in terms of the general surface stress hypothesis that hydrostatic pressure forces newly formed wall to expand in a particular direction. What distinguishes one type of organism from another is the regions of the cell where new wall growth occurs. For several classes of organisms, the pattern of growth deduced from the shape agrees with biochemical, morphological and physiological studies. Gram-negative rods, as typified by Escherichia coli, have a morphology that may be explained in several ways by this general hypothesis.
In the present paper, the morphological, autoradiographic and biochemical data concerning E. coli are reviewed. Thirteen models are considered; there is reason to reject most of them but one model that includes two others appears more satisfactory. All the models conform to the biophysical principles that it is impossible to turn over stress-bearing peptidoglycan without shape change and that growth of the sacculus requires the introduction of new oligosaccharides and their covalent linkage before cleavage of stress-bearing bonds. The model that best accounts for the experimental data assumes that, while addition of peptidoglycan all over the cell is possible and does occur, the effective surface tension is higher (and therefore the rate of wall growth is very small) in old poles than on the sides, and very much lower at the developing division sites (where the rate of wall growth is high). It is shown that, when the hydrostatic pressure is constant throughout the cell and during a large part of the cell cycle, changes in the biochemical mechanism of wall growth which correspond to a decrease in surface tension would lead to an invagination of the stress-bearing wall. An additional mechanism may, however, be needed for final closure and for the splitting of the last few covalent bonds holding the two nascent cells together.
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Spatial Resolution of Autoradiograms of Rod-shaped Organisms
More LessIsotope-containing rod-shaped bacteria approximate a line-segment source for purposes of autoradiography. This is because the width is small and bacteria are usually cylindrically symmetric. For this reason published work has only classified the silver grains longitudinally, independent of the transverse position. While the theoretical integral distribution from a variety of geometric shapes has been calculated, the line-segment has been overlooked because long narrow objects well separated from other sources occur infrequently in eukaryotic biology. This oversight is corrected here. In addition, a simple program for line-segments and ways to combine distributions from separate line-segment sources is presented. Some typical distributions are depicted. The program is used elsewhere in the analysis of wall growth patterns of bacteria.
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Effect of Plasma-membrane Phospholipid Unsaturation on Solute Transport into Saccharomyces cerevisiae NCYC 366
More LessA comparison was made of kinetics of solute accumulation by Saccharomyces cerevisiae NCYC 366 grown anaerobically under conditions that lead to enrichment of the plasma membrane with ergosterol and either oleyl or linoleyl residues. Values for K T and V max were identical for accumulation of l-asparagine, l-glutamine, H2PO4 2-, Ca2+ and SO4 2-, while for accumulation of d-glucose, the value differed slightly but not significantly. Values for K T for accumulation of l-lysine, by both the low-and high-affinity systems, decreased when oleyl residues were replaced by linoleyl residues. Under these conditions, V max values for the high-affinity system decreased while that for the low-affinity system increased. An Arrhenius plot for accumulation of lysine by the high-affinity system revealed a discontinuity when membranes were enriched in linoleyl residues. However, no discontinuity was evident on plots of lysine accumulation when membranes were enriched in oleyl residues. Similar plots for accumulation of l-asparagine, which was used as a control, showed that substitution of linoleyl for oleyl residues significantly raised the transition temperature, but had little effect on the activation energy at temperatures below the discontinuity. When palmitoleyl residues were incorporated into the yeast plasma membrane, the K T value for l-lysine accumulation by the high-affinity system was hardly altered, although the V max value was lowered, as compared with organisms with membranes enriched in oleyl residues. Replacement of oleyl by palmitoleyl residues lowered both the K T and V max values for accumulation of l-asparagine. A modified statistical method is described for calculating confidence limits for transition points on Arrhenius plots.
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Accumulation of L-Asparagine by Saccharomyces cerevisiae X-2180
More LessAt concentrations up to 1 mm, l-asparagine was accumulated by Saccharomyces cerevisiae X-2180 at 30 °C against a concentration gradient. Values for K T and V max were, respectively, 3.·5 × 10−4 M and 33 nmol (mg dry wt)−1 min−1. At concentrations below 0·1 mm, a convex curve was obtained on a Woolf-Hofstee plot, possibly indicating the presence of two l-asparagine-binding sites. Autoradiograms of extracts of organisms that had accumulated labelled l-asparagine revealed only one spot with an R F value identical with that of l-asparagine. Four mutant strains lacking the ability to synthesize the general amino acid permease system grew and accumulated l-asparagine at similar rates to the parent. The rate of accumulation of l-asparagine from a 0·2 mm solution was greatest at pH 4·5, with the decrease in accumulation rate greater at values below than above 4·5. l-Glutamine, l-histidine, l-methionine, l-threonine and l-tryptophan caused appreciable inhibition of the rate of l-asparagine accumulation. With the exception of l-methionine, the inhibition caused by these amino acids was competitive. Several other amino acids, including d-asparagine and l-aspartic acid, caused little or no inhibition of l-asparagine accumulation.
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Accumulation of Intracellular Solutes by Two Filamentous Fungi in Response to Growth at Low Steady State Osmotic Potential
More LessGlycerol and the solutes used to control the steady state osmotic potential of the medium, either glucose, glucose and fructose, or KC1, accumulated in the hyphae of Penicillium chrysogenum and Chrysosporium fastidium as the external potential was lowered. When the osmoticum was KC1, nearly all the observed hyphal osmotic potential of P. chrysogenum could be accounted for from the measured solute concentrations, but the proportion decreased when a glucose osmoticum was used, to 56% in the case of P. chrysogenum and to 65% for C. fastidium, when grown at an external potential of − 10 MPa. Higher polyols were present in considerable quantity in both species but did not appear to be involved in osmotic adjustment.
Potassium was the predominant cation in both species when grown on glucose, but chloride was insufficient to maintain electroneutrality. The potassium to sodium ratios were considerably higher in C. fastidium than P. chrysogenum, and inversely related to the external potential in the former species only.
Glycerol and glucose were present in greater amounts at the margin than in older parts of large colonies of P. chrysogenum. High turgor potentials were apparently maintained throughout these large colonies which meant that the observed osmotic potential could not be accounted for by the solutes measured in older parts of the colony.
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Effect of Osmotic Shock on Some Intracellular Solutes in Two Filamentous Fungi
More LessHypoosmotic and hyperosmotic shock experiments confirmed the importance for osmoregulation in Penicillium chrysogenum and Chrysosporium fastidium of glycerol and the osmoticum used in the medium. Growth ceased following both types of shock treatment for a time, depending on the magnitude of the shock and the species. Regrowth following hypoosmotic shock took place some distance behind burst tips. Following hyperosmotic shock, growth was initiated by branching at the apex, but if the magnitude of the shock was greater than 10 MPa, it did not occur in either species although osmotic adjustment was observed. Shock experiments did not implicate the higher polyols in osmotic adjustment. Hypoosmotic shock to C. fastidium resulted in a rapid loss of all internal solutes, while decrease in glycerol in P. chrysogenum following this treatment was more gradual. Transfer of C. fastidium, a species which does not grow on media of high salt concentration, to isoosmotic KCl did not result in loss of turgor although growth was inhibited. Glucose was lost from the hyphae but K+ and Cl− were taken up.
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Growth and Accumulation of Solutes by Phytophthora cinnamomi and Other Lower Fungi in Response to Changes in External Osmotic Potential
More LessProline and the solute used to control the osmotic potential of the medium, either sucrose or KCl, accumulated in the hyphae of Phytophthora cinnamomi as the steady state external potential deceased. Proline also accumulated in three other lower fungi. Very little polyol was detected in any of the species analysed. Proline was lost following hypoosmotic shock and accumulated after hyperosmotic shock in P. cinnamomi. The time taken for growth to resume after shock treatment was variable, but less than 5 h. Potassium was the predominant cation in hyphae grown on sucrose but chloride was insufficient to maintain electroneutrality. The potassium to sodium ratio followed a similar pattern to the radial growth rate with respect to external potential, with maxima at a potential slightly below the highest potential tested. When the osmoticum was sucrose, the calculated internal potential was 20% greater than the observed osmotic potential, but when the osmoticum was KCl, the calculated potential was too high by a factor of more than two, probably indicating error in assumptions concerning the location of ions within the hyphae.
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Thiamin Accumulation and Growth Inhibition in Yeasts
More LessThiamin caused depression of growth, a marked decrease in cellular vitamin B6 content and cytochrome oxidase activity in Saccharomyces yeasts growing in a vitamin B6-free medium under aerobic conditions but had practically no effect in Kluyveromyces, Schizosaccharomyces and Candida spp. Pyridoxine added concomitantly with thiamin permitted the thiaminsensitive yeasts to grow normally with increased activity of cytochrome oxidase. δ-Aminolaevulinate also caused the increase in cytochrome oxidase activity but growth was only partially improved by the addition of this precursor of haem biosynthesis. These phenomena were similar to those found previously in Saccharomyces carlsbergensis strain 4228 (ATCC 9080) ( Nakamura et al., 1981 ). Thiamin-sensitive yeasts accumulated thiamin more than 24-fold when compared with the thiamin-insensitive cells. Thiamin transported into the thiamin-sensitive yeasts was recovered only in the non-esterified form. Thiamin added to the growth medium was also accumulated by growing cells of the thiamin-sensitive yeasts especially during the lag and early exponential phases of growth. Pyridoxine did not affect either thiamin accumulation or the intracellular form of the transported thiamin.
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Semi-continuous and Continuous Production of Aspergillus niger Spores in Submerged Liquid Culture
More LessSporulation of Aspergillus niger was induced in continuous tower fermenters by restricting growth with nitrate limitation. Semi-continuous production of spores was achieved in a single-stage fermentation by alternating full-nutrient and nitrogen-deficient media to the culture. Continuous spore production was obtained in a two-stage fermentation system using the first stage for the growth of vegetative mycelium under optimum conditions and the second, larger, stage for sporulation induction in a constant nitrogen-deficient environment. Spores were produced from much simplified sporulation structures. Using a new sporulation index (Ω), which relates spore numbers produced to the quantity of substrate utilized, the average production efficiency of the two-stage continuous system was shown to be more than twice that of the semi-continuous production system. The tower fermenter was shown to be ideal for controlling organism morphology, thus providing conditions which allowed the development of continuous fungal sporulation.
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Changes in Intracellular pH Accompanying Chemoreception in the Plasmodia of Physarum polycephalum
More LessA new method for measuring intracellular pH, employing the intrinsic fluorescent pigments of the plasmodia of the myxomycete Physarum polycephalum, was used to study the role of pH in chemotactic transduction in the plasmodia. The cell became acidified following stimulation with the attractants alanine, glucose, galactose and maltose when their concentrations exceeded the respective thresholds of chemoreception and taxis. The degree of cell acidification paralleled the relaxing tendency in tension generation. A non-metabolizable attractant. 2-deoxyglucose, also acidified the cell. However, the repellent salts NaCl, KCl and CaCl2 did not change the intracellular pH. Our results suggest that the effects of attractants are mediated by intracellular pH, while the effects of repellents are transduced by ATP as reported previously.
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Inhibition of Nicotinic Acid and Nicotinamide Uptake into Bordetella pertussis by Structural Analogues
More Less3-Pyridine-carboxaldehyde and 3-pyridine-aldoxime were effective and specific inhibitors of the uptake of both nicotinic acid (NA) and nicotinamide (ND) by Bordetella pertussis, although neither compound inhibited the growth of the bacteria in liquid medium or the oxidation of glutamate by washed suspensions. In contrast, the following pyridine derivatives did not inhibit uptake of NA or ND: iso-NA, iso-ND, isoniazid, 6-amino-NA and 6-amino-ND, 3-acetyl-pyridine, 3-pyridyl-acetic acid, N,N-diethyl-ND and 3-pyridine-sulphonic acid. 3- Pyridyl-carbinol was inhibitory, but less so than the first listed compounds.
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Differentiation of Saccharomyces cerevisiae at Slow Growth Rates in Glucose-limited Chemostat Culture
More LessSaccharomyces cerevisiae was grown in a glucose-limited chemostat at dilution rates equivalent to mass doubling times of 2 to 16 h. Phase contrast microscopy revealed that cultures with mass doubling times greater than 4·5 h contained two sub-populations of cells, phase-dark and phase-light. The latter were shown to be predominantly unbudded daughter cells, which were not proliferating, and had properties similar to those of stationary phase cells in batch culture. Phase-dark cells were equivalent to cells growing exponentially in batch culture. The results suggest that the formation of phase-light cells represents cell cycle specific differentiation into stationary phase. The consequences of this phenomenon for the interpretation of results obtained with S. cerevisiae in the chemostat are discussed.
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Localization of Carpophore Initiation in Coprinus congregatus
More LessThe differentiation of mushroom primordia in young mycelia of Coprinus congregatus is a localized event: the mycelium is not uniformly competent to differentiate, or even to be induced, in all areas. A narrow zone corresponding to the 24 h growth of the youngest hyphae is the only area capable of receiving the obligatory light induction and of eventually differentiating into primordia. The inducible zone migrates outwards with the peripheral hyphae as the mycelium expands and previously competent zones become inactive and non-inducible. Light induction of a zone fixes the zone of differentiation in space and prevents subsequent zones from becoming induced. The light receptor system appears to involve two independent processes. One is activated by very low levels of light, fixes the zone in space and prevents subsequent zones from becoming induced, but does not permit differentiation. A second, requiring higher light levels, triggers development of the primordia. The induction of a responsive zone can occur at 20 °C, but actual development requires 25 °C.
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The Role of Laccase in Carpophore Initiation in Coprinus congregatus
More LessThe mycelium of the basidiomycete Coprinus congregatus is not uniformly competent to differentiate. Differentiation occurs only in localized areas of young mycelia. Studies on laccase activities from whole mycelium extracts suggest a correlation between overall laccase levels and the development of mushroom primordia. However, studies of higher resolution on the localized areas of developmental competence, under a variety of light, nutrient and temperature regimes, indicate that laccase is probably not involved in the actual development of the primordia. If laccase is involved in C. congregatus differentiation, it is only at the initial, light-requiring, step.
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- Short Communications
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Facile in vivo Transfer of Mutations Between the Bacillus subtilis Chromosome and a Plasmid Harbouring Homologous DNA
More LessTransformation of Bacillus subtilis strains carrying the point mutations spoIIA42 or spoIIA69 by a plasmid, pHM2, carrying the spoIIA + allele gave occasional recombinants in which the plasmid had acquired the chromosomal mutation. Transformation of a spo + strain by these derivative plasmids gave occasional recombinants in which the chromosome of the recipient had acquired the plasmid-borne spoIIA mutation. Both types of behaviour were recE + dependent. Mutations spoIIA42 and spoIIA69 fell into a single complementation group.
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Rapid Tissue Culture Method for Detection of Mycoplasma hyorhinis
More LessAn easy, rapid tissue culture method for detection of Mycoplasma hyorhinis is described. The organism induces morphological changes in mink S + L - cells. This effect was not observed in eight other animal cell lines infected by M. hyorhinis and it did not occur in the mink cells infected with six other strains of mycoplasma. This cell system should be useful in research laboratories which do not have other standard techniques available for monitoring the presence of M. hyorhinis.
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- Taxonomy
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Fatty Acid Composition of Some Mycolic Acid-containing Coryneform Bacteria
More LessThe fatty acid profiles of 74 strains of mycolic acid-containing coryneform bacteria were examined by gas-liquid chromatography. All of the strains contained major amounts of straightchain and monounsaturated fatty acids although some also possessed substantial amounts of 10-methyloctadecanoic acid. Iso- and anteiso-branched acids were not present. Five distinct fatty acid patterns were evident: (i) Corynebacterium diphtheriae, C. pseudotuberculosis and C. ulcerans strains contained major amounts of hexadecanoic and hexadecenoic acids; (ii) C. glutamicum, C. xerosis and related saprophytic and animal-associated strains, predominantly hexadecanoic and octadecenoic acids; (iii) C. bovis, major amounts of octadecenoic and 10-methyloctadecanoic acids; (iv) C. mycetoides, significant amounts of heptadecanoic acid as well as hexadecanoic and octadecenoic acids; and (v) strains related to Rhodococcus possessed significant quantities of 10-methyloctadecanoic acid in addition to straight-chain and monounsaturated acids.
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