- Volume 114, Issue 2, 1979
Volume 114, Issue 2, 1979
- Biochemistry
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Characterization of the Autolytic Enzymes of Clostridium perfringens
More LessClostridium perfringens and isolated walls of this organism autolysed rapidly when incubated in buffer at pH 7·0 with the release of free-reducing groups but no N-terminal amino acids. The predominant autolytic enzyme was an endo-β-N-acetylglucosaminidase, and an endo-β-N-acetylmuramidase was also present. The autolytic enzymes could be solubilized by extraction of the organisms with 5 m-LiC1 and would then subsequently bind to and rapidly lyse walls of Micrococcus luteus and, more slowly, formamide-extracted walls of C. perfringens and walls of Bacillus subtilis. Lysis of C. perfringens walls by these extracted enzymes could not be demonstrated.
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A Biochemical Explanation for Lipid Accumulation in Candida 107 and Other Oleaginous Micro-organisms
More LessThe biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mm). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor.
The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependentisocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, themetabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomesarrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase whichconverts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate.
Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.
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Factors Affecting Aflatoxin Production by Aspergillus parasiticus in a Chemically Defined Medium
More LessSummary: The optimum levels of sucrose, (NH4)2SO4, MgSO4, KH2PO4 and ZnSO4 for aflatoxin production in a chemically defined medium have been established. The last two were found to be essential for fungal growth and aflatoxin production. The effect of various carbon sources on aflatoxin production was tested using the defined medium. Asparagine was found to be essential for aflatoxin production. Very little aflatoxin was produced in the absence of asparagine with any of the other inorganic nitrogen sources tested. Supplementation with yeast extract, Casamino acids, Casitone and peptone increased the aflatoxin yield, but omission of asparagine led to decreased aflatoxin yields even when complex nitrogen sources were present. Asparagine could be replaced by aspartic acid or alanine.
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Cell Wall-associated 1,4-β-d-Xylanase in Cryptococcus albidus var. aerius: in situ Characterization of the Activity
More LessSummary: 1,4-β-d-Xylanase (1,4-β-d-xylan xylanohydrolase; EC 3.2.1.8) has been detected in both cell-free extracts and culture fluids of the yeast Cryptococcus albidus var. aerius grown on glucose as the only carbon source. Mild acid treatment of whole cells proved that the enzyme was extracellularly located. The activity remained almost completely linked to the wall after cell breakage, only being liberated in the presence of salt at high concentration. After release, the enzyme became very unstable and so has been characterized in situ in permeabilized cells. The maximum production took place at the beginning of the exponential growth phase. The optimum pH and temperature for activity were 5·0 and 40 °C, respectively. The enzyme degraded xylan and xylo-oligosides by an endo-splitting mechanism giving xylobiose, xylotriose and xylose as the main end-products. Activation energy and kinetic constants for xylan degradation were determined. Several metal ions such as Ag+ and Hg2+ inhibited the enzyme. The possible function of this endo-xylanase in Cr. albidus var. aerius is discussed.
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- Development And Structure
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Phenotypes of Double Conidiation Mutants of Aspergillus nidulans
More LessA series of strains, doubly mutant at conidiation loci, have been made. The phenotypes of these strains reflected the epistasy of earlier blocking mutants over later ones and confirmed the order of gene sequence predicted from the phenotypes of single mutants. Oligosporogenous mutants gave complex interactions, especially between brl and med mutants. These results indicated that (i) gene action overlapped in time, (ii) several parts of the conidial apparatus were interchangeable and (iii) nuclei leaving the vesicle were not irreversibly programmed. Structures produced by mutants were reminiscent of the conidial apparatus of other Aspergillus species and of related genera.
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Factors Controlling the Formation of Akinetes Adjacent to Heterocysts in the Cyanobacterium Cylindrospermum licheniforme Kütz
More LessSubstances which stimulate the formation of akinetes (spores) in Cylindrospermum licheniforme Ktz. are secreted into a phosphate-free sporulation medium by filaments of that cyanobacterium. One such substance, purified from the centrifugal supernatant fluid of sporulating cultures, initiated sporulation in a phosphate-containing culture medium. Certain amino acids, particularly tryptophan, and calcium glucuronate also strongly stimulated sporulation. Acetylene and ethylene were inhibitory. No significant effect of cyclic nucleotides was observed. The addition of 12·5 to 15 % (v/v) H2 stimulated sporulation in air or under CO2/O2/Ar (0.1:19.9:80, by vol.) by up to 2·5-fold, but did not significantly affect the reduction of acetylene by intact filaments. Thus, the stimulation of sporulation by hydrogen was not mediated by an effect on the fixation of nitrogen. Hydrogen uptake in a cell-free suspension derived from whole filaments was detected manometrically, with phenazine methosulphate as electron acceptor, at a rate of 5·8 mol H2 (mg chlorophyll a)−1 h−1. The uptake hydrogenase activity derived from isolated heterocysts accounted for 84 ± 2 % of the uptake hydrogenase activity from whole filaments, whereas no activity was detected in a fraction derived from the vegetative cells.The formation of the pattern consisting of spores contiguous with heterocysts may be controlled by either, or a combination of, (i) a sporulation-stimulatory substance, if that substance is synthesized solely in heterocysts, or (ii) some substance the concentration of which may be controlled by hydrogen assimilated by heterocysts.
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Isolation and Characterization of a Substance which Stimulates the Formation of Akinetes in the Cyanobacterium Cylindrospermum licheniforme Kütz
More LessA substance which at low concentration (less than 0·3 μm) stimulated the formation of akinetes in Cylindrospermum licheniforme Kütz. was purified from the centrifugal supernatant fluid of sporulating cultures. High resolution mass spectrometry showed that the chemical formula of the substance is C7H5OSN. Other peaks of high intensity found at m/e values of 123 and 96 in the mass spectrum were produced by loss of CO from the molecular ion, and by additional loss of HCN. Proton nuclear magnetic resonance spectroscopy of the substance showed a complex of peaks in the region of 𝛿= 7·19 to 7·29 p.p.m. Peaks in the infrared absorption spectrum were attributable to methylene C-H bonds and to C = S and cyclic -CO-NH-groups. The most probable structure consistent with the above findings embodies two fused, five-membered rings, one of which is a lactarn and the other of which has a thioketone group.
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- Ecology
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Plasmids, Biological Properties and Efficacy of Nitrogen Fixation in Rhizobium japonicum Strains Indigenous to Alkaline Soils
More LessPlasmids were isolated from strains of Rhizobium japonicum, predominantly serogroup 135, obtained from soybean nodules collected at 15 sites in Nebraska, U.S.A. In addition to their serotype, these strains were indistinguishable from R. japonicum strain 311b135 in growth rate, sensitivity to phage Rhj781, antibiotic sensitivities, general colony characteristics and rates of nitrogen fixation per plant. All strains occupied soil habitats with similar characteristics, including a high pH (7·2 to 8·3), relatively high conductivity (0·04 to 0·32 mS), relatively high sodium saturation (0·32 to 12·7%), low iron content (3·2 to 14·8 p.p.m.) and low manganese content (5·1 to 18·7 p.p.m.). However, agarose gel electrophoresis analysis of plasmids enabled subdivision of these extra-slow-growing strains into four groups on the basis of differences in plasmid number and size. These strains carried combinations of two or more of four plasmids, ranging in mass from 49 to 118 megadaltons and comprising approximately 20% of the total DNA per cell. Biological and symbiotic data, along with plasmid analysis, were useful in identifying a wild-type strain (RJ23A) that shows potential as a soybean inoculant in alkaline soils.
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- Genetics And Molecular Biology
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Methionine Transport in Salmonella typhimurium: Evidence for at Least One Low-affinity Transport System
More LessSummary: The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a K m of 0·3 μ m for l-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0·3 to 1·1 %) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent K m values for methionine of 24 μ m and approximately 1·8 mm. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.
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radE, a New Radiation-sensitive Locus in Dictyostelium discoideum
More LessDictyostelium discoideum strain M28, which has been used widely in genetic studies, was found to carry a radiation-sensitive mutation. This allele, termed rad-100, was recessive in heterozygous diploids and mapped in linkage group III. Complementation analysis and survival studies on strains carrying rad-100 suggested that this allele defines a new radiation-sensitive locus in D. discoideum, and this locus has been designated radE. radE strains were moderately sensitive to ultraviolet light (D 10 90 J m−2) and slightly sensitive to 137Cs gamma rays (D 10 255 krad). radE strains also exhibited increased sensitivity to killing by N-methyl-N′-nitro-N-nitrosoguanidine but not by other alkylating agents such as ethyl methanesulphonate or methyl methanesulphonate. The frequency of spontaneous methanol-resistant (acrA) mutants was approximately the same in cultures of radE and radE + strains. However, when amoebae of these strains were irradiated with ultraviolet light, the frequency of induced mutants was significantly lower in cultures of the radE strain. Furthermore, when amoebae of wild-type strain NC4 were plated in the presence of caffeine after ultraviolet-irradiation, the survival curves were very similar to the curves obtained for amoebae of radE strains in the presence or in the absence of caffeine. These results suggest that the radE100 mutation and caffeine interfere with an error-prone DNA repair pathway in D. discoideum.
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Characterization and Persistence of Actinophage RP2 Isolated from Streptomyces rimosus ATCC 10970
More LessSummary: While searching for true lysogens among oxytetracycline-producing Streptomyces rimosus strains, free phage particles were detected and isolated from a liquid culture of S. rimosus ATCC 10970 (R7). The actinophage, designated RP2, appears to be a typical temperate DNA phage producing turbid plaques on the sensitive strain S. rimosus R6. Electron microscopic examination of RP2 lysates showed that it belongs to group B of Bradley's morphological classification. The rate of RP2 adsorption at 28 °C appeared to be low. The length of the latent period was about 6h and the average burst size about 120 phage particles.
The lysogenic nature of the host-virus system described was established on the basis ofthe following characteristics: spontaneous lysis frequency of 2 × 10−6 per cell, resistance tocuring with phage-specific antiserum, spontaneous curing frequency of less than 0·05% yoand immunity to superinfection with the homologous phage. Clear-plaque mutants of RP2,which failed to lysogenize sensitive cultures, arose at a frequency of 10−5
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Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens
More LessSummary: Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3·8 × 10−6 to 1·7 × 10−7 per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.
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Studies on Recently Isolated Cultures of Methicillin-resistant Staphylococcus aureus
More LessSummary: Of 19 recently isolated cultures of raethicillin-resistant Staphylococcus aureus, 18 showed inducible low-level resistance to minocycline, 15 showed high-level resistance to streptomycin, and 4 showed resistance to low levels of streptomycin. Two cultures produced yellow pigment and may have been derived in vivo by loss of a gene(s) determining orange pigment.
Treatment of three cultures with serial exposures to N-methyl-N′-nitro-N-nitrosoguanidine resulted in a widening of phage typing pattern that included all reactions in group I, the great majority in group 111, but none in group 11. The widening in phage lysis was possibly due to the elimination of defective prophages. Transfer of tetracycline resistance occurred from 12 out of the 19 cultures to a recipient in mixed culture; this transfer required either Ca2+ or Mg2+, was abolished by citrate, and enhanced by high cell density. It was probablymediated by defective bacteriophages.
No evidence was obtained for the occurrence of recombination within the methicillinresistant clone in nature. Eleven methicillin-resistant cultures stored for at least 5 years on agar slopes at 20 °C had all lost this resistance at high frequency.
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Relationship of Group P1 Plasmids Revealed by Heteroduplex Experiments: RP1, RP4, R68 and RK2 Are Identical
More LessSummary: The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7·8% portion of incomplete reannealing. This ‘heterologous region’ could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with λ phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.
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Identification of Different Sites of Expression for spo Loci by Transformation of Bacillus subtilis
More LessAsporogenous mutants of Bacillus subtilis were rendered capable of forming heat-resistant spores by transformation with wild-type (spo +) DNA at, or near, the start of sporulation. For several mutants up to about 50 % of the colonies derived from heat-resistant spores, formed as a result of the transformation, remained genetically asporogenous (spo). This was thought to indicate that the genome of the mother cell, but not that of the forespore, was transformed to spo + and that correct expression of the spo locus in the mother cell was sufficient for spore formation. At the end of the process the mother cell was destroyed, leaving a mature heat-resistant spore that was genetically asporogenous. It is concluded that the loci spoIIID, spoIVA, spoVB and spoVE are expressed in the mother cell. For one mutant more than 99 % of the colonies derived from heat-resistant spores were genetically spo +. It is concluded that the locus involved, spoVA, had to be expressed in the forespore. Thus different sporulation-specific loci are expressed in the mother cell and in the forespore. The loci expressed in the mother cell are expressed in one cell type so that another cell type, the forespore, can develop into a heat-resistant spore.
Other unselected donor markers could be introduced into the recipient during transformationprovided high concentrations of DNA were used. The frequency of congressionwas the same for spo survivors as for spo+ survivors. This implies that there was nocorrelation between the DNA strand into which the selected spo+ and the unselected donor markers integrated.
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Isolation and Partial Characterization of Four Plasmids from Antibiotic-resistant Thermophilic Bacilli
More LessSummary: Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4·9 × 106 (7·0 kilobases) and pAB118B of molecular weight 3·0 × 106 (4·3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2·9 × 106 (4·14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2·5 × 106 (3·57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis 168 (Trp−) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.
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Evidence that p-Fluorophenylalanine has a Direct Effect on Tubulin in Aspergillus nidulans
More LessThree temperature-sensitive alleles of benA (benA11, 17 and 21) confer resistance to growth inhibition by p-fluorophenylalanine (FPA). FPA resistance cosegregates with the benA gene. Two back-mutations in benA which cause loss of temperature sensitivity cause loss of FPA resistance, and two indirect suppressors of benA temperature sensitivity also cause FPA resistance to be lost. These results indicate that FPA resistance is an intrinsic property of the benA mutations. The intracellular phenylalanine concentrations of these strains are normal as is their ability to take up phenylalanine from the medium. We conclude that FPA must inhibit growth and cause non-disjunction by a direct effect on the polymerization of tubulin.
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- Medical Microbiology
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Variation in the Virulence of Strains of Mycoplasma pulmonis Related to Susceptibility to Killing by Macrophages in vivo
More LessThe virulence of five strains of Mycoplasma pulmonis, as judged by their ability to survive in the respiratory tract and induce pneumonia in CBA mice, was related to the ability of viable organisms to persist in the peritoneal cavity. This appeared to be the result of differences in the ability of the strains to resist killing by peritoneal macrophages in vivo. It is suggested that resistance to phagocytosis by macrophages is an important determinant of virulence for M. pulmonis.
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Variations in Surface Protein Composition Associated with Virulence Properties in Opacity Types of Neisseria gonorrhoeae
More LessThe biological properties of a series of opacity variants of Neisseria gonorrhoeae P9 have been examined. A novel protein, designated protein II* (mol. wt 28850), was identified within the set of heat-modifiable surface proteins previously reported. All variants producing extra outer membrane proteins were less sensitive to the bactericidal action of serum than the prototype transparent strain, with protein IIa* (mol. wt 28500) being associated with increased resistance. The production of a different protein, protein II* (mol. wt 29000), was correlated with resistance to low molecular weight antimicrobial agents (penicillin, fusidic acid, Cu2+, Zn2+). Increased adhesion to human buccal epithelial cells was demonstrated in all variants that produced extra surface proteins. These variants did not show increased binding to hexyl- and phenyl-substituted Sepharose gels suggesting that hydrophobic interaction was not responsible for their cohesive properties. The prototype strain lacking additional proteins demonstrated the greatest binding to erythrocytes. indicating that adhesion to buccal cells and red blood cells is mediated by different mechanisms. One variant producing protein IIa* showed increased association with leukocytes, whereas another producing protein IIb* showed decreased association with leukocytes. These results show that the heat-modifiable surface proteins are important virulence attributes of the gonococcus: this must be considered in the selection of strains for vaccine trials.
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The Effect of Reducing and Other Agents on the Motility of Treponema pallidum in an Acellular Culture Medium
More LessThe maintenance of Treponema pallidum motility was investigated in an acellular medium based on T. pallidum immobilization test medium. The acellular medium contained cysteine, glutathione, thioglycollate and dithiothreitol as reducing agents and had a redox potential of −275±25mV at pH 7·3. In an atmosphere containing 3% O2, motile treponemes survived four times longer when calf serum and bovine serum albumin were added to the medium. The selective omission of glutathione and, particularly, thioglycollate prolonged the survival of motile treponemes almost fivefold. In addition, stored medium, in which thioglycollate had become inactive, sustained motile treponemes for longer than did freshly prepared medium. Thus, thioglycollate is toxic for the organisms. It may be omitted from the medium because low redox potentials can be achieved without it.
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