- Volume 130, Issue 4, 1984
Volume 130, Issue 4, 1984
- Biochemistry
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Production of Bacteriolytic Enzymes and Degradation of Bacterial Cell Walls During Growth of Agaricus bisporus on Bacillus subtilis
More LessThe mechanism by which the mycelium of Agaricus bisporus can use Bacillus subtilis as sole carbon and nitrogen source was investigated. During fungal growth more than 80% of the muramic acid and diaminopimelic acid residues in the bacterial walls disappeared from the microbial biomass and appeared as soluble glycopeptides. Culture supernatants dissolved purified walls of B. subtilis, and bacteriolytic enzymes were obtained by ion-exchange chromatography of these supernatants. The main bacteriolytic enzyme activity was found to be a β-N-acetylmuramidase. Soluble bacterial wall fragments were partially purified by chromatography of the culture fluids. The chemical properties of these fragments were consistent with their having been produced by β-N-acetylmuramidase action on the peptidoglycan of the B. subtilis cell wall.
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A Model for the Common Control of Enzymes of Ethanolamine Catabolism in Escherichia coli
More LessBy varying the composition of the growth medium and the genotype of the bacterial strain, five isoenzymes of CoA-dependent aldehyde dehydrogenase could be detected in Escherichia coli. Two isoenzymes (A, mol. wt 520000; and B, mol. wt 370000) were produced only in the presence of ethanolamine and vitamin (or coenzyme) B12 (‘inducible isoenzyme’), whereas the other three isoenzymes (C, mol. wt 900000; D, mol. wt 120000; and E, mol. wt 720000) were produced only in the absence of ethanolamine and vitamin B12 (‘repressible isoenzymes’). Partial purification and characterization of these isoenzymes revealed strong similarities, with respect to pH optima and substrate affinities, between isoenzymes within either of the two classes, but significant differences between the two classes. Mutant studies demonstrated that the relationships between the isoenzymes and between CoA-dependent aldehyde dehydrogenase and ethanolamine ammonia-lyase are both structural and regulatory in nature, and a two-operon model is proposed to account for the common control of the enzymes of ethanolamine catabolism in E. coli.
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Lipid-bound Saccharides Containing Glucose and Galactose in Agrobacterium tumefaciens
More LessThe incubation of an enzyme preparation of Agrobacterium tumefaciens with UDP-[14C]Glc led to the formation of radioactive substances soluble in chloroform/methanol/water (1 : 1 : 0·3, by vol.). These substances had properties similar to the polyprenol diphosphate saccharides. The lipid moiety appears to be an unsaturated polyprenol. Mild acid hydrolysis of the substances liberated five water-soluble products which were separated by TLC and characterized by borohydride reduction-acid hydrolysis, partial acid hydrolysis, methylation analysis, TLC and paper chromatography and paper electrophoresis. The products behaved like: galactose; the disaccharide Glc(β1-3)Gal; the tetrasaccharide Glc(β1-4)Glc(β1-4)Glc(β1-3)Gal; an octasaccharide and a pyruvylated octasaccharide. The last two substances were compared by paper electrophoresis, TLC and methylation analysis with an octasaccharide obtained by the action of a hydrolytic enzyme on the exopolysaccharide of Agrobacterium tumefaciens. The octasaccharide and the pyruvylated octasaccharide from the lipid behaved like those obtained by enzyme action on the A. tumefaciens exopolysaccharide. These results suggest that the lipid-bound saccharides are involved in the biosynthesis of the extracellular polysaccharides.
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Purification, Properties and Regulation of Glutamine Synthetase from Nitrobacter agilis
More LessGlutamine synthetase (EC 6·3·1·2) was purified about 430-fold from the nitrifying bacterium Nitrobacter agilis by affinity chromatography on Blue Sepharose CL-6B and gel filtration on Sepharose 4B. The enzyme (apparent mol. wt 700000), which consisted of 12 subunits, each of mol. wt 58000, required a divalent cation for both biosynthetic and transferase activities. Regulation of glutamine synthetase both by a feedback inhibition involving amino acids and by an adenylylation/deadenylylation mechanism was studied. The enzyme was highly adenylylated (75-90%) in cell free extracts from cells grown on nitrite as the sole source of nitrogen. The adenylylated form of the enzyme could be deadenylylated by treatment with snake venom phos-phodiesterase. An isoactivity pH of 7·4 was recorded for glutamine synthetase.
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NAD+ - and NADP+-Dependent Glutamate Dehydrogenases in Nitrobacter agilis
More LessTwo isoenzymes of glutamate dehydrogenase located in the cytoplasmic fraction of Nitrobacter agilis were specific for NAD+ and NADP+, respectively. The NAD+-dependent enzyme functioned in both directions, i.e. amination and deamination, whereas the NADP+ enzyme was primarily for the amination of 2-oxoglutarate to glutamate. The NADP+ enzyme was purified 52-fold (free of the NAD+ enzyme) by affinity chromatography on 2′,5′-ADP Sepharose 4B, and some of its properties studied. Substrate activation of the amination reaction of the NADP+ enzyme was observed with NH+ 4 and NADPH. A comparison is made of the properties of the purified NADP+ enzyme from Nitrobacter agilis and Nitrosomonas europaea. The possible roles of two isoenzymes of glutamate dehydrogenase in Nitrobacter agilis are discussed.
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Thioredoxin as a Modulator of Glucose-6-phosphate Dehydrogenase in a N2-Fixing Cyanobacterium
More LessGlucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme involved in fixed carbon dissimilation in photosynthetic micro-organisms; in heterocystous cyanobacteria it may also be implicated in the supply of reductant to nitrogenase. In crude cell-free extracts of the N2-fixing cyanobacterium Anabaena variabilis G6PDH activity was reversibly deactivated by the thiol agent dithiothreitol in the presence of a low molecular weight protein (12000mol. wt). Glucose 6-phosphate reversed deactivation when added at high concentration, or prevented deactivation if added with the thiol. NADP+, which, like glucose 6-phosphate, is a G6PDH substrate, also deactivated the enzyme; deactivation was reversed or prevented by adding glucose 6-phosphate or glutamine. Purified thioredoxin from Anabaena cylindrica, at very low concentrations (2 nm), deactivated purified G6PDH in a manner identical to that observed when crude extracts were used in the presence of dithiothreitol. Glutathione did not affect the enzyme.
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Purification and Molecular and Catalytic Properties of Phosphoribulokinase from the Cyanobacterium Chlorogloeopsis fritschii
More LessPhosphoribulokinase (ATP:d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was purified from the cyanobacterium Chlorogloeopsis fritschii. The enzyme had a molecular weight of 230000, as determined by gel filtration, and a pH optimum of 8·6. Divalent cations were essential for activity, maximal activity being supported by Mg2+, while Mn2+, Ca2+ and Co2+ were less effective. AMP, ADP, phosphoenolpyruvate, aspartate and malate inhibited enzyme activity completely at 1 mm. No effects on phosphoribulokinase activity were observed with NAD, NADH, NADP or NADPH at up to 10 m m or with glyoxylate at up to 20 mm. The enzyme was activated in semi-purified extracts by the addition of dithiothreitol and reduced glutathione. SDS-PAGE of SDS-dissociated enzyme revealed only one polypeptide band of molecular weight 40000. This suggests that C. fritschii phosphoribulokinase is a hexamer consisting of six subunits of identical size.
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Relationships Between Proton Extrusion and Fluxes of Ammonium Ions and Organic Acids in Penicillium cyclopium
More LessAcidification observed in cultures of Penicillium cyclopium was mostly due to the extrusion of protons into the medium. In the absence of NH+ 4 protons were extruded together with citrate. The citric acid thus produced underwent a dynamic equilibrium of continuous efflux and uptake, which was shifted towards the latter at decreasing glucose concentrations. In cultures supplied with NH+ 4 and glucose a stoichiometric coupling of H+ excretion with NH+ 4 uptake was observed. If the protons excreted were not absorbed by external buffers (e.g. tartrate ions), the pH of the medium dropped to below 2, thereby exceeding the capacity of the cells to maintain their internal pH. The decrease of intracellular pH was accompanied by the cessation of NH+ 4 uptake, H+ extrusion and growth. In the presence of tartrate ions the NH+ 4/H+ exchange proceeded until the glucose concentration of the medium dropped below 25 mm. Here, the external pH was kept above 3, without seriously affecting the intracellular pH. After termination of H+ extrusion an appreciable amount of NH+ 4 was taken up together with hydrogentartrate anions, thereby causing a further acidification of the medium, which was finally reversed due to the uptake of tartaric acid in the late phase of growth.
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- Development And Structure
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Control of Wall Band Splitting in Streptococcus faecalis
More LessComputer reconstructions of 659 and 1325 whole mounted, shadowed cells, randomly chosen from cultures of Streptococcus faecalis undergoing balanced growth and doubling in mass every 83 min and 30 min, respectively, were used to analyse the cell cycle. The size limits and duration of phases of the cell cycle were estimated by applying a method previously described by the authors, details of which are given here to allow others to use the method. Deeply constricted cells whose primary septal radius, R s, was less than or equal to 0·18 μm were considered as belonging to an E-phase ending the cell cycle. The statistical parameters of these E-phase cells were used to calculate the mean and coefficient of variation of dividing cells. These latter values, in turn, predicted the moments of the total population well enough so that the method's assumptions were judged adequately satisfied. Therefore, the method was considered applicable to other phases and sub-phases of the cell cycle of these two cultures. The E-phase cells were further classified as having either 0, 1 or 2 secondary growth zones, allowing us to calculate the percentage of newborn cells without growth zones. In the slow-growing cells. 69% of the cells arose with no growth zone. On the other hand, in more rapidly growing cells 16% of the cells or less arose with no growth zone. Our calculations showed that they could exist without a growth zone for only 2 and 0·1 min, respectively. We also classified cells as possessing a ‘birth site’if the volume between the two daughter bands was greater than 0, but less than 0·06 μm 3. From the statistical properties of such cells with new growth zones, the mean pole time, W, was estimated. We also estimated W from the size of cells in E-phase. The major conclusion is that the pole time is only slightly greater than the mass doubling time at both growth rates. Since DNA synthesis in S. faecalis takes longer (C = 50 to 52 min) than the mass doubling time in rich medium (30 min), a new round of chromosome replication must be initiated before the old round of synthesis is completed (dichotomous replication). Consequently, wall band splitting and initiation of chromosome replication do not occur simultaneously. It was also concluded that the cell initiates wall band splitting, resulting in pole formation and cell division, when the growth zones cannot function rapidly enough to allow the increase of surface area required to accommodate continuing production of cytoplasm.
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Sporulation of Bacillus sphaericus 2297: an Electron Microscope Study of Crystal-like Inclusion Biogenesis and Toxicity to Mosquito Larvae
More LessSporulation of Bacillus sphaericus strain 2297 in a synchronous liquid culture was studied by electron microscopy. The t 0 of sporulation occurred 7 h after the beginning of the lag phase. Crystal-like inclusions first appeared at t 2 and reached their final size between t 5 and t 6. The release of the spore/inclusion complex occurred at about t 15 (22 h after inoculation).
Toxicity against Culex pipiens larvae was related to sporulation and appeared during the early stages of sporulation. The LC50 (24 h) decreased about 105-fold between t 0–2 and t 7, in correlation with the formation of crystalline inclusions. Heat resistance of spores appeared later than toxicity.
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Synthesis of the Exosporium During Sporulation of Bacillus cereus
More LessTechniques of antigenic analysis were used to examine the synthesis of the exosporium during sporulation of Bacillus cereus. An antiserum to a soluble extract of exosporium from mature spores was used to analyse extracts of cells from various stages of the Bacillus life cycle by the technique of one-dimensional immunoelectrophoresis. Exosporium antigens were absent from vegetative cell extracts and were first detected in extracts of cells at stage III of sporulation, where they appeared simultaneously in the soluble and particulate cellular fractions. The results indicate that the exosporium contains antigens which are spore specific.
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- Ecology
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The Follicular Distribution and Abundance of Resident Bacteria on Human Skin
More LessA cryostat sectioning procedure was used to determine quantitative viable counts of microorganisms both on the surface and in successive layers of human cadaver skin biopsies. Also, using a previously described xenograft model, we investigated the dependence of microorganisms on the presence of sebaceous glands by using full thickness (1520 mm) and split thickness (06 mm, ensuring sebaceous gland exclusion) human skin. Our results show substantial variation in the distribution and abundance of skin bacteria, even amongst biopsies from the same cadaver. In general, propionibacteria were distributed within a narrow band at varying depths beneath the skin surface whereas staphylococci were more broadly distributed. The importance of this with respect to topically applied antiseptics and antimicrobial agents is indicated. The xenograft studies demonstrated that propionibacteria were dependent on the presence of sebaceous glands whereas staphylococci were not.
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The Microbial Ecology of Pilosebaceous Units Isolated from Human Skin
More LessA method allowing isolation and microbiological analysis of individual pilosebaceous units (follicles) was used to study biopsies of back skin obtained from volunteer acne vulgaris patients. The main microbial groups isolated were members of the genera Propionibacterium, Staphylococcus and Pityrosporum. The incidence (and mean density) of these organisms in 140 normal follicles was 12% (2·6 × 105 per follicle), 4% (5·5 ×103 per follicle) and 13% (102 per follicle) respectively. Colonized follicles were not distributed evenly amongst the subjects studied. The results are analysed and discussed from an ecological standpoint.
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Effects of Sedimentation and Light Intensity on Mat-forming Oscillatoriaceae with Particular Reference to Microcoleus lyngbyaceus Gomont
More LessThe distribution and abundance of mat-forming Oscillatoriaceae were investigated at a siltdepositing freshwater site in England over a period of 4 years. Maximum growth occurred during April and May and experiments showed that mat buried by fine sediment could regain the surface by gliding movements. The number of trichomes reaching the surface was light-dependent but chemotactic responses were also apparent. Gliding rates of 1–2 μs−1 were recorded and the trichomes moved through the sediment as a left-handed screw with a pitch of 60°. A surface illumination of 1 klx (4 W m−2) was optimal for mat formation. Light intensities exceeding 2·5 klx produced a photophobic response resulting in lateral trichome migration to regions of lower surface illumination when these were provided. Migration into the sediment was not observed in the surface intensity range 0–10 klx. The onset of light saturation measured by the 14CO2 method was 3 klx for Microcoleus lyngbyaceus, close to the optimum for mat development. Experiments employing 14CO2 in the presence of sulphide and absence of oxygen suggested that the organisms were adapted to an aerobic environment.
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- Genetics And Molecular Biology
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Peptide Antibiotics and Sporulation: Induction of Sporulation in Asporogenous and Peptide-negative Mutants of Bacillus brevis
More LessMutants of Bacillus brevis ATCC 8185 were isolated which were unable to produce detectable amounts of either tyrocidine or linear gramicidin, or both peptide antibiotics. Tyrocidine-negative mutants (BM5, BM21, BM44) sporulated normally. Gramicidin-negative mutants (BM2, BM24) were oligosporogenous, and mutants unable to produce both peptides (S18, S19) were asporogenous. Addition of tyrocidine and/or gramicidin to asporogenous mutants in rich medium did not stimulate sporulation. However, these mutants formed normal spores after being transferred to nitrogen-free medium and upon the addition of tyrocidine. It was demonstrated that nutrient broth has a suppressive effect on tyrocidine-induced sporulation of S18. The tyrocidine-negative mutant BM44, sporogenous in rich medium, could sporulate under nitrogen deprivation only if supplemented with tyrocidine. The significance of the peptide antibiotics for a regulatory role in sporogenesis of B. brevis is discussed.
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Cloning of Sporulation Gene spoIIC in Bacillus subtilis
More LessSpecialized transducing phages pllspoC and ϕ105spoIIC, carrying the Bacillus subtilis sporulation gene spoIIC, were constructed by the prophage transformation method. An EcoRI fragment (2·4 MDal) carrying the spoIIC gene was isolated from the ϕ105spoIIC genome and recloned into the EcoRI site of plasmid pUB110. The recombinant plasmids corrected the sporulation defect of a Spo− Rec− host, but slightly inhibited the sporulation of a Spo+ Rec− host.
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Effect of UV Irradiation on Macromolecular Synthesis and Colony Formation in Bacteroides fragilis
More LessIrradiation of Bacteroides fragilis cells with far-UV light resulted in the immediate, rapid and extensive degradation of DNA which continued for 40 to 60 min after irradiation. During the degradation phase, DNA synthesis was decreased but was never totally inhibited. DNA degradation after irradiation was inhibited by chloramphenicol and caffeine. DNA synthesis in irradiated cells was reduced by chloramphenicol but resumed after 100 min at the same exponential rate as in irradiated cells without chloramphenicol. Irradiated cells continued to synthesize DNA for 40 min in the presence of caffeine but after this time DNA synthesis was completely inhibited and never recovered. RNA and protein synthesis were decreased by UV irradiation and the degree of inhibition was proportional to the UV dose. Colony formation was not affected immediately by UV irradiation and continued for a dose-dependent period before inhibition. There was an inverse relationship between UV dose and inhibition of colony formation which occurred sooner in cells irradiated with lower doses of UV light. The characteristics of DNA synthesis in B. fragilis cells after UV irradiation differ from those in wild-type Escherichia coli cells, where DNA synthesis is stopped immediately by UV irradiation, but resemble those in E. coli recA mutant cells where extensive degradation occurs following UV irradiation.
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Chromosomal Loci Associated with Antibiotic Hypersensitivity in Pulmonary Isolates of Pseudomonas aeruginosa
More Less492a and 492c were two strains of Pseudomonas aeruginosa isolated from the sputum of a patient with cystic fibrosis. The strains were closely related but expressed different antibiograms. 492c was hypersensitive (10-100 times more sensitive than 492a) to the β-lactam antibiotics carbenicillin, methicillin, flucloxacillin, mecillinam and cefuroxime and the non-β-lactam, nalidixic acid. 492c also showed enhanced sensitivity (48 times more sensitive than 492a) to chloramphenicol, trimethoprim and novobiocin. 492a and PAO8 expressed similar levels of antibiotic resistance, except for trimethoprim, to which 492a was five times more sensitive than PAO8. Two genes associated with antibiotic hypersensitivity were mapped in the 30 min region of the chromosome, by means of R68.45-mediated plate matings between a Leu− mutant of 492c and PAO8, followed by transductional analysis using phage F116L. The first of these genes, blsA1 was closely linked to nalBand in a PAO background, was associated with hypersensitivity to the β-lactams and a moderate increase in sensitivity to chloramphenicol, trimethoprim, nalidixic acid and novobiocin. A further increase in sensitivity to the latter three antibiotics was associated with the second gene, tpsA1, which mapped between ser-3 and hisV. This gene could also be transferred to PAO from 492a, thus 492c could have arisen from 492a in vivo following a single chromosomal mutation at the blsA locus. Isolation of a blsA mutant of PAO969 provided further evidence for this theory.
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Location and Direction of Transcription of the ptsH and ptsI Genes on the Escherichia coli K12 Genome
More LessRecombinant plasmids were constructed that carried various fragments of the DNA specifying the Escherichia coli genes ptsH and (part of) ptsI, the genes for the common components of the phosphoenolpyruvate : sugar phosphotransferase. Expression of plasmid-specified functions in minicells showed that ptsH and ptsI were transcribed clockwise. Most of the transcription of ptsI was from the ptsH promoter, but some was from a second site within or after ptsH.
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The Effect of Inhibitors of DNA Repair on the Genetic Instability of Streptomyces cattleya
V. E. Coyne, K. Usdin and R. KirbyVarious streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss. The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations. The frequency of instability can be enhanced by UV irradiation. Two major repair systems have been found in Escherichia coli: the ‘error-free’ system which is inhibited by caffeine and the ‘error-prone’ system which is inhibited by arsenite. Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development. Some evidence was also found for the presence of an arsenite inhibitable UV repair system. The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S. cattleya. The arsenite system may be implicated in the repair of such events. Genetic instability was also induced by single strand breaks in DNA caused by 32P.
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- Immunology
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Demonstration of the Common Antigens of Clostridium botulinum, C. sporogenes and C. novyi by an Enzyme-linked Immunosorbent Assay and Electroblot Transfer
More LessEDTA extracts were prepared from whole cells of 16 strains of Clostridium botulinum (types A-E), 6 strains of C. novyi (types A-D) and 3 strains of C. sporogenes. They were reacted in an enzyme-linked immunosorbent assay (ELISA) with antisera raised against whole, UV-killed cells of C. sporogenes and C. novyi type A. Results showed significant cross-reactions between C. sporogenes antiserum and the C. botulinum type A (three out of four strains), proteolytic type B (all strains) and one type E strain, and between C. novyi type A antiserum and C. botulinum types C and D. All the C. sporogenes and C. novyi strains reacted with their homologous antiserum; these two species showed no cross-reactions. All the reactions were investigated further by running the EDTA extracts on SDS-polyacrylamide gels. The separated molecules were electro-phoretically transferred to nitrocellulose membranes, reacted with antiserum and complexes visualized with horseradish peroxidase conjugate reagents. Only those extracts that reacted significantly in the ELISA gave a pattern of cross-reactive antigen bands and the number of bands and intensity of stain closely paralleled the strength of the ELISA reaction.
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- Pathogenicity And Medical Microbiology
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The Conditioning Role of Saliva in Streptococcal Attachment to Hydroxyapatite Surfaces
A. Abbott and M. L. HayesThe solution properties of saliva and its role in conditioning both the substrate and the bacterial surface have been investigated with regard to the attachment of oral streptococci to hydroxyapatite surfaces. Saliva from eight subjects was used and the attachment of three organisms, Streptococcus mutans strains FA-1 (serotype b) and KPSK2 (serotype c) and S. sanguis T175-1, was studied. An adsorbed salivary layer on a hydroxyapatite surface substantially reduced the affinity of the organisms for the surface. Adsorbed saliva on the bacterial surfaces, however, tended to increase the organisms affinity for saliva-coated apatite. The source of saliva was important in determining the extent of inhibition of attachment. The data indicated that the negatively charged and hydrophilic nature of salivary conditioning films was important in controlling bacterial adsorption to hydroxyapatite. The results also suggested that hydrophobic salivas could promote binding of the more hydrophobic bacteria known to be early colonizers of the teeth.
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Antibiotic Susceptibility of Legionella pneumophila Philadelphia-1 in Cultured Guinea-Pig Peritoneal Macrophages
More LessThe effect of antimicrobial agents on the intracellular multiplication of Legionella pneumophila in cultured guinea-pig peritoneal macrophages was measured. Beta-lactam antibiotics at concentrations 5 to 400 times the MIC in vitro did not inhibit the intracellular growth of the organism. Gentamicin inhibited the growth considerably but failed to eliminate the organism from the phagocytic mixture. Chloramphenicol or tetracycline at 10 μg ml−1 (40 or 5 times the MIC in vitro respectively) did not eliminate the organism. At a higher concentration (30 μg ml−1), however, these drugs eliminated the bacterium from the mixture. Only erythromycin and rifampin were effective in killing the organism at very low concentration (1 μg ml−1). Intracellular multiplication of L. pneumophila was observed clearly by light microscopy using Wright-Giemsa staining.
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Surface Antigens of Intact Aspergillus fumigatus Mycelium: their Localization Using Radiolabelled Protein A as Marker
More LessSummary: Isotopically-labelled Protein A from Staphylococcus aureus was used as a marker to quantify binding of IgG molecules to surface antigens of Aspergillus fumigatus mycelium. The IgG class antibodies were obtained from rabbits inoculated with mycelial fractions and from patients suffering from aspergillosis and aspergillus-related diseases. The highest incorporation of label was obtained with an antiserum from rabbits inoculated with A. fumigatus wall material. Antibodies raised to other antigenic fractions of A. fumigatus and antibodies from patients infected with aspergillus gave lower levels of incorporation of Protein A. In competitive binding experiments, pre-incubation of antibodies with partially purified aspergillus antigens depressed subsequent binding to the mycelial surface by 20-30% of that of the control values. Low molecular weight disaccharides and oligosaccharides were without effect in this system. Preincubation of A. fumigatus with lectins having specificities for defined sugar residues did not reduce subsequent antigen/antibody interaction. Treatment of the mycelial surface with certain proteolytic or polysaccharolytic enzymes led to a decrease in antibody binding, while pretreatment of A. fumigatus with the hydrolytic enzyme mixture of Trichoderma harzianum culture filtrate gave increased antibody binding. Aspergillus species showed different susceptibilities to enzyme action and their surface structures could be differentiated from A. fumigatus on this basis. These differences were not obvious in direct binding experiments where antibodies raised to A. fumigatus wall bound with equal facility to antigenic sites located on the walls of other Aspergillusspecies.
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Preparative Isoelectric Focusing of Immunologically Reactive Components of Aspergillus fumigatus Mycelium
More LessA water-soluble mycelial extract of Aspergillus fumigatus has been fractionated by preparative isoelectric focusing using carrier ampholytes in a layer of granulated gel. The separated components were located by staining paper prints from the gel. Within a narrow pH range of 2·5 units, multiple protein bands were visualized with Coomassie Brilliant Blue G. Periodate-Schiff-positive material was generally associated with the major protein zones. When these fractions were eluted the total recovery, calculated on the basis of protein and carbohydrate analyses of the isolated fractions, varied between 20 and 60% of the applied material. Low recoveries were associated with low recoveries of protein; recoveries of carbohydrate were higher and less variable. The immunological activity and specificity of the eluted fractions were assessed in an enzyme-linked immunosorbent assay for the detection of IgG antibodies to A. fumigatus.
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Polycationic Polypeptides: a Possible Model for the Penetration-enhancing factor in the Invasion of Host Cells by Toxoplasma gondii
More LessThe effect of polycationic polypeptides (polylysine, polyarginine and polyhistidine) on the invasion of mammalian cells and plant protoplasts by Toxoplasma gondii was studied. In JM cells, a human lymphoblastoid cell line with T cell characteristics, all polycationic polypeptides used increased the invasion rate in a concentration-dependent manner. This effect and the morphological changes revealed by electron microscopy resembled the action of the penetration-enhancing factor previously described by E. Lycke and co-workers. Plant protoplasts of Catharanthus roseus, which are resistant to T. gondii invasion, showed the same morphological changes in the presence of polycationic polypeptides as observed for JM cells, but were not invaded.
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- Physiology And Growth
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Mutants of Escherichia coli with Altered Hydrogenase Activity
More LessMutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2. They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.
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Heterocyst Differentiation in Cylindrospermum licheniforme: Studies on the Role of Transcriptio
More LessHeterocysts of the cyanobacterium Cylindrospermum licheniforme occur at the ends of the filaments. These cells, specialized for aerobic N2 fixation, synchronously differentiate after the fragmentation of filaments grown in a medium free of combined nitrogen. This study has examined the role of transcription during and after heterocyst differentiation. Autoradiography of intact filaments pre-labelled for 3 h with [3H]uracil revealed that RNA synthesis occurred at similar rates in vegetative cells, developing (pro)heterocysts and mature heterocysts. Since mature heterocysts no longer divide and contain a similar amount of DNA as vegetative cells, transcription must continue under conditions where replication no longer occurs. Rifampicin, when added to fragmented filaments at concentrations that reduced RNA synthesis by 95% in all cell types, inhibited proheterocyst formation but not the final morphological step of differentiation (pore plug deposition). However, chloramphenicol blocked all stages of heterocyst differentiation. Since the average mRNA half-life was 29 min, and the rifampicin-insensitive stage lasted 3–6 h, the later stages of differentiation may depend on long-lived mRNAs.
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Betaine Stimulation of Vitamin B12 Biosynthesis in Pseudomonas denitrificans May Be Mediated by an Increase in Activity of δ-Aminolaevulinic Acid Synthase
More LessThe stimulation by betaine of vitamin B12 and extracellular coproporphyrin production in the industrial species Pseudomonas denitrificans GY57/2 was confirmed using a replacement culture technique; biosynthesis of haem was also found to be stimulated. In the absence of betaine, δ-aminolaevulinic acid synthase decreased in specific activity from time zero. In the presence of betaine, the enzyme activity increased markedly after 3 h and then decreased after exhaustion of extracellular betaine. The peak specific activity of δ-aminolaevulinic acid synthase increased in a dose-dependent manner. The enzyme was inhibited by haem. δ-Aminolaevulinic acid dehydratase was present in considerable excess over δ-aminolaevulinic acid synthase under all conditions studied and its specific activity was not affected by the addition of betaine. Our tentative conclusion is that the betaine effect is exerted via some positive effect on δ-aminolaevulinic acid synthase, the initial, rate-limiting and very labile enzyme of porphyrin and corrin biosynthesis.
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Nitrogen-fixing Growth in Continuous Culture of a Strain of Rhizobium sp. Isolated from Stem Nodules on Sesbania rostrata
More LessA strain of Rhizobium sp. (ORS571), isolated from stem nodules on Sesbania rostrata, was grown as a N2-fixing continuous culture for up to five weeks (> 80 culture replacements). Under optimum conditions of O2 supply [< 9 m dissolved O2, 100160 nmol O2 (mg dry wt)−1 min−1], N2 fixation rates were 300400 nmol N2 (mg dry wt)−1 h−1 and nitrogenase activity in culture samples reached 2000 nmol C2H4 (mg dry wt)−1 h−1, the highest values yet recorded for any Rhizobium species. Other properties of the continuous cultures are described and a comparison is made with previous reports of N2 fixation by cultures of Rhizobium spp. Significant features were the greater tolerance to O2 of ORS571 and the retention of fixed N in the cells of this strain.
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Acquisition of Aspartase Activity in Rhizobium leguminosarum WU235
More LessRhizobium leguminosarum WU235 expresses aspartase (EC 4.3.1.1) when grown on aspartate or asparagine as the sole carbon source, but not on glucose or fumarate. Cells grown on glucose plus aspartate, or fumarate plus aspartate, do not express aspartase. Although these results are reminiscent of catabolite control of an inducible enzyme, induction of aspartase cannot be demonstrated in this strain. Aspartase-producing cells synthesize the enzyme after repeated subculture on glucose plus NH4Cl. Cells grown in glucose plus NH4Cl and plated onto aspartate produce different colony sizes; the larger (0·1% of the total) express aspartase, while the smaller do not. At dilutions sufficient to exclude the large aspartase-producing colonies, a single-sized, aspartase-negative colony was found initially. Such colonies later developed papillae or became cluster colonies; aspartase was produced with papillae formation. The aspartase producing strains were shown, by analysis of native plasmids and periplasmic proteins and by the use of antibiotic resistant strains, to be derived from the parental type. The data suggest that strain WU235 is unable to produce aspartase unless a mutation occurs which leads to constitutive enzyme synthesis. The significance of these observations for studies claiming catabolite repression in Rhizobium is discussed.
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Light-microscopic Visualization of F and Type 1 Pili
More LessSummary: Methods for the direct visualization of F and type 1 pili of Escherichia coli in the light microscope are described. The method for visualizing F pili is based on the specific adsorption of fluorescent dye-labelled RNA phages to F pili. The best results were obtained with MS2 phages labelled with rhodamine B. Semi-quantitative determination of the amount of F pili is possible. Type 1 pili can be visualized rapidly and specifically by indirect immunofluorescence. Other structures on the cell surface are neither detected by, nor interfere with these assays. By using different fluorescent dyes the two methods can be combined and both F and type 1 pili can be determined in the same sample.
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F and Type 1 Piliation of Escherichia coli
More LessSummary: F and type 1 piliation of various Escherichia coli K12 Hfr and F+ strains was re-examined by using a new visualization assay. In anaerobic cultures in rich media, bacteria were well F piliated throughout all growth phases. In aerobic cultures in rich media, F piliation reached a maximum in the mid-exponential phase. The yield of pili was up to 15 mg 1−1. In aerated cultures. F pili disappeared in the late exponential phase. In rich media F pili were on average 10-20 μm long; in synthetic media they were an order of magnitude shorter, and less numerous. Addition of metabolic poisons (NaCN, NaN3, Na3 AsO4, phenylethyl alcohol) and starving the bacteria caused rapid disappearance of F pili under aerobic conditions, but had no influence on F piliation under anaerobic conditions. Type 1 piliation was not influenced by these drugs or by an alteration of growth conditions.
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- Systematics
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Menaquinone Composition in the Classification of Actinomadura and Related Taxa
More LessMenaquinones were the only isoprenoid quinones found in 40 strains representing 19 species of Actinomadura, Microbispora rosea, Microtetraspora glauca, Microtetraspora niveoalba, Nocardiopsis dassonvillei and Streptomyces somaliensis. The Nocardiopsis dassonvillei strains had a characteristic profile consisting of major amounts of di-, tetra-, hexa- and octahydromenaquinones with ten isoprene units and were thereby distinguished from all of the other test strains which contained complex mixtures of hydrogenated menaquinones with nine isoprene units. The latter strains were divided into three groups based upon the predominant isoprenologue detected: (i) Actinomadura citrea, A. coerulea, A. helvata, A. libanotica, A. livida, A. luteofluorescens, A. madurae, A. malachitica, A. verrucosospora and Streptomyces somaliensis contained hexahydrogenated menaquinones with nine isoprene units; (ii) A. pelletieri and A. spadix contained octahydrogenated menaquinones with nine isoprene units and (iii) ‘ coeruleoviolacea’, A. ferruginea, A. pusilla, A. roseola, A. roseoviolacea, A. rubra, A. salmonea, A. spiralis, Microbispora rosea, Microtetraspora glauca, and M. niveoalba contained tetrahydrogenated menaquinones with nine isoprene units. The results show that menaquinone profiles are of value both in generic and sub-generic classifications of Actinomadura. They also suggest that some species of Microbispora and Microtetraspora are closely related to Actinomadura, a result in line with other trends in the taxonomy of these organisms.
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- Short Communication
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The Effects of Pectin and Plant Root Surface Carbohydrates on Encystment and Development of Phytophthora cinnamomi Zoospores
More LessCitrus pectin induces highly synchronous differentiation in populations of Phytophthora cinnamomi zoospores in the absence of a host plant. This action is independent of the presence of Ca2+ in the zoospore suspension medium. Water-soluble, ethanol-insoluble material present on the surface of the plant roots also induces differentiation of P. cinnamomi zoospore populations with the same time course as pectin. It is proposed that this material provides the natural recognition signal which induces P. cinnamomi zoospores to differentiate, and that pectin is sufficiently similar in structure for it to mimic its action.
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DNA Restriction Endonuclease Analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG
More LessDNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.
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