- Volume 132, Issue 10, 1986
Volume 132, Issue 10, 1986
- Biochemistry
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Urease Activity of the Cyanobacterium Anabaena cylindrica
More LessSUMMARY: The cyanobacterium Anabaena cylindrica was found to have nickel-dependent urease activity. Addition of nickel to a nickel-free culture led to recovery of urease activity. This was not prevented by chloramphenicol. Cell-free extracts of nickel-depleted cells did not regain activity upon addition of Ni2+. Enzyme synthesis was constitutive; similar urease activities occurred in urea-, NO− 3- or NH3-grown cultures or those grown under N2-fixing conditions. The urease activity had hyperbolic kinetics, with an apparent K m with respect to urea of 1.3 ± 0.6 (SD) mM. Enzyme activity was enhanced up to two fold by MgSO4 and MnSO4 at pH 7.5. At 10 mM-urea, cells hydrolysed urea and excreted NH3 in excess of biosynthetic requirements.
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- Development And Structure
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Inhibition of Reproduction of Phytophthora by the Calmodulin-interacting Compounds Trifluoperazine and Ophiobolin A
More LessSUMMARY: Sporangium production in Phytophthora palmivora was inhibited by trifluoperazine (TFP) and ophiobolin A. TFP was inhibitory to a similar extent to cultures both in light and in darkness, although the stimulatory effect of calcium and the inhibitory effect of EGTA were much greater in the dark. Sporangium and oospore production in P. cactorum were inhibited by TFP. Thus development of both asexual and sexual reproductive structures involves a calcium/calmodulin interaction.
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Changes in the Plasma Membrane of Regenerating Protoplasts of Candida albicans as Revealed by Freeze-fracture Electron Microscopy
More LessSUMMARY: Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.
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Methionine Requirement of Sporulation in a Streptomyces fradiae Mutant
More LessSUMMARY: When an Arg-mutant of Streptomyces fradiae was grown on sucrose-nitrate agar supplemented with L-citrulline, it produced only non-sporulating aerial mycelia. Sporulation could be induced by adding 0.1 to 10.0 mM-L-methionine or 0.01 mM-cyanocobalamin; several other compounds including homocysteine, other amino acids, vitamins and precursors of nucleic acids were ineffective. Growth of the mutant did not differ significantly with or without methionine. The mutant was more sensitive to L-norleucine (a methionine analogue) than the parent strain. Spontaneous mutants, resistant to this analogue, were obtained. They all sporulated well without exogenously added methionine. In medium-exchange experiments, methionine was required for sporulation only after 48 h cultivation. The phenomenon reported here is most probably based on partially impaired methionine biosynthesis caused by an as yet uncharacterized non-auxotrophic mutation, since Arg+ revertants retained their methionine requirement for sporulation, and were also sensitive to L-norleucine.
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- Ecology
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Microbiostasis by Nutrient Deficiency Shown in Natural and Synthetic Soils
More LessSUMMARY: The germination pattern of six species of fungi on synthetic soil containing antibiotic-producing or non-antibiotic-producing micro-organisms was similar to that on natural soil. Sterilization by autoclaving destroyed the fungistatic effect of both natural and synthetic soils. Mixed microorganisms were more effective than bacteria, actinomycetes or fungi alone in inducing fungistasis in synthetic soil. The percentage germination of Exserohilum rostratum and Bipolaris maydis on both natural and synthetic soils increased with increase in the proportion of silica sand added. Bacteriostasis, actinostasis and fungistasis occurred concurrently in the synthetic soil, which also induced lysis of mycelia of Neurospora tetrasperma. Preincubation on natural or synthetic soil rendered nutrient agarose blocks incapable of supporting germination of nutrient-dependent fungi without reducing their ability to support germination of nutrient-independent fungi. Individual groups of micro-organisms were not as effective as mixed micro-organisms in causing diffusion of nutrients from agarose blocks to synthetic soil.
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- Genetics And Molecular Biology
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The arcABC Operon Required for Fermentative Growth of Pseudomonas aeruginosa on Arginine: Tn5-751-assisted Cloning and Localization of Structural Genes
More LessSUMMARY: Pseudomonas aeruginosa is able to utilize L-arginine as the energy source for growth under anaerobic, nitrate-free conditions. Mutations in the chromosomal arcABC gene cluster specifying the inducible arginine deiminase pathway enzymes abolish fermentative growth on arginine. From two different arc::Tn 5-751 insertion mutants of P. aeruginosa recombinant plasmids have been derived which carry a resistance marker of transposon Tn5-751 plus flanking parts of the arc region. These recombinant plasmids served to reconstruct in vitro the functional arcABC cluster on a 5.6 kb fragment, which was inserted into the broad-host-range vector pKT240. In P. aeruginosa this 5.6 kb segment complemented arcABC mutations in trans and contained the control region necessary in cis for arc enzyme induction by oxygen limitation and arginine. The results of subcloning experiments and transcriptional lacZ fusions, the polarity of transposon insertions and the effect of external promoters led to the conclusion that the structural genes arcA (for arginine deiminase), arcB (for catabolic ornithine carbamoyltransferase) and arcC (for carbamate kinase) are contiguous and transcribed in the same direction. Thus, the arcABC cluster appears to have the characteristics of an operon. In Escherichia coli the cloned arcABC genes were expressed at low, non-inducible levels; strong vector promoters enhanced arc expression up to 100-fold. This indicates that transcriptional initiation at the arc promoter(s) is poor in E. coli.
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Effect of Alkylating Agents on the Expression of Inducible Genes of Escherichia coli
More LessSUMMARY: Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.
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R-Plasmid-mediated Chromosome Mobilization in Bordetella pertussis
More LessSUMMARY: Antibiotic-resistant and auxotrophic mutants of Bordetella pertussis were isolated. These were used as recipients for the uptake from Escherichia coli of broad-host-range R plasmids R68.45, RP1, and RP1 and RP4 carrying transposons Tn501 and Tn7 respectively. B. pertussis transconjugants from these crosses were used as donors to mobilize StrR, NalR, thr + and gly + chromosomal markers to B. pertussis or to B. parapertussis recipient strains. The frequency of plasmid transfer varied and depended on the donor and recipient strains used. Differences in chromosome mobilization frequencies of individual markers were observed and appeared to depend on the presence or absence of transposons Tn501 and Tn7 on the plasmid. Linkage was detected between the gly + and NalR markers.
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Expression and Purification of Glutamine Synthetase Cloned from Bacteroides fragilis
More LessSUMMARY: A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B. fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified: it had an apparent subunit M r of approximately 75000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.
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Methylation of Spore DNA in Bacillus coagulans Strain 26
More LessSUMMARY: The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes. A significant fraction of the GATC sequences (dam target) in spore DNA contain N 6-methyladenine, a modification that is lacking during the vegetative phase. From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred. Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase.
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Bacteriophages Fo lac h, SR, SF: Phages Which Adsorb to Pili Encoded by Plasmids of the S-Complex
SUMMARY: Phage Fo lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid Fo lac. A host range mutant, phage Fo lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS:: Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage Fo lac h. Phages Fo lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.
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A Restriction Map of Naphthalene Catabolic Plasmid pWW60-1 and the Location of Some of Its Catabolic Genes
More LessSUMMARY: A restriction map for the 87 kbp IncP9 naphthalene catabolic plasmid pWW60-1 is presented. Transposon mutants were obtained using direct Tn5 insertion or using an indirect method involving the intermediate formation of unstable cointegrates between RP4 and pWW60-1. Insertions which affected expression of the early enzymes of the pathway (naphthalene to salicylate) were separated from inserts which affected expression of salicylate hydroxylase (nahG) or catechol 2,3-oxygenase (nahH). nahABC were cloned on a contiguous region of the plasmid on either a 6.9 kbp HindIII fragment (HE) or a 5.7 kbp XhoI fragment (XD). nahGH were cloned on a region situated about 30 kbp from nahABC on an XhoI fragment (XC), but nahH was only expressed on the corresponding but smaller XhoI fragments from two derivative plasmids of pWW60-1 with a deletion in that region. The detailed restriction map of nahH shows no similarities with the restriction maps of the genes for catechol 2,3-oxygenases from TOL plasmids, although the cloned gene did hybridize with genes for catechol 2,3-oxygenase from two different TOL plasmids. The organization of the catabolic genes on pWW60-1 suggests a separation into two operons as also described for the naphthalene catabolic plasmid NAH7 (alternatively called pIG7), but the relative directions of their transcription differs from that in NAH7.
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Restriction Mapping and Close Relationship of the DNA of Streptomyces erythraeus Phages 121 and SE-5
More LessSUMMARY: The biological properties and genome structure of two actinophages, 121 and SE-5, infecting Streptomyces erythraeus were characterized. They had the same host range (limited to S. erythraeus) and similar DNA G + C contents (around 60 mol %). Restriction maps of their genomes also showed many similarities. The close relationship between the two phages was confirmed by DNA hybridization experiments: large parts of their genomes were homologous, except for a segment in the middle of the map, where no hybridization was detected.
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- Pathogenicity And Medical Microbiology
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Ampicillin Resistance and Penicillin-binding Proteins of Haemophilus influenzae
More LessSUMMARY: Penicillin-binding protein (PBP) alterations have been associated with non-β-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 μg ml−1 for the susceptible isolates at an inoculum of 100000 c.f.u. when tested by broth dilution, and was 0.5 μg ml−1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 μg ml−1 when tested by broth dilution, and from 1.5 to 16 μg ml−1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics. These results suggest (a) that care should be taken in interpreting changes in PBP profiles for species that demonstrate variability such as H. influenzae, and (b) that the decreased binding affinity of PBP 5 is a consistent finding associated with multiple ampicillin-resistant wild-type isolates.
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Adherence to Uroepithelial Cells of Providencia stuartii Isolated from the Catheterized Urinary Tract
More LessSUMMARY: The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (108 bacteria per 105 cells). Washed UEC, retained on 8 μm pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.
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Sucrose-dependent Cell Adherence and Cariogenicity of Serotype c Streptococcus mutans
More LessSUMMARY: Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.
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Demonstration of Shared Antigenic Determinants between Streptococcus mutans BHT Cell Membrane, Human Heart Tissue and Myosin Using Monoclonal Antibodies to S. mutans
More LessSUMMARY: Monoclonal antibodies (MAb) raised to intact Streptococcus mutans P-4 cells (serotype e) were used to demonstrate the presence of shared antigenic determinant(s) between S. mutans BHT (serotype b) cell membranes and human heart tissue. MAb binding to both BHT membrane and human heart tissue was demonstrated by ELISA. Common antigens were identified by immunoblot analysis following separation of BHT membrane components and human heart antigens by SDS-PAGE. MAb 22C4 recognized three polypeptides from the BHT membrane preparation, having molecular masses of 42, 56 and 85 kDa. MAb 22C4 also recognized an 85 kDa component and a 200 kDa component from human heart tissue. MAb D159 was specific for a single 82 kDa polypeptide in BHT membrane, and also bound to two high molecular mass components in human heart (165 and 200 kDa). When both MAb D159 and 22C4 were first absorbed with S. mutans P-4 cells, subsequent reactivity to the aforementioned BHT membrane components was inhibited, indicating that these cross-reactive components are found in S. mutans P-4 as well as in S. mutans BHT micro-organisms. Competitive binding analysis showed that both MAb D159 and MAb 22C4 bound to myosin, indicating that S. mutans BHT membrane, human heart tissue and myosin share at least one immunodeterminant. This indicates that myosin could be the cross-reactive tissue component in human heart.
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A Scanning Electron Microscope Study of the Effect of an Enterotoxin from Clostridium perfringens 8-6 on Mice of Different Ages
More LessSUMMARY: Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy. Two distinct types of damage were observed, both of which could be correlated with animal age. Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits. We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age.
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- Physiology And Growth
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Pentose Metabolism in Rhizobium leguminosarum MNF300 and in Cowpea Rhizobium NGR234
More LessSUMMARY: L-Arabinose is broken down by Rhizobium leguminosarum MNF300 via 2-oxoglutarate semialdehyde. Enzyme activities in cells grown on succinate, mannitol or arabinose indicated much greater modulation of arabinonate dehydratase, 2-keto-3-deoxyarabinonate dehydratase and 2-oxoglutarate semialdehyde dehydrogenase than of arabinose dehydrogenase or of arabinono-γ-lactonase. In cowpea Rhizobium NGR234, all the enzymes of L-arabinose metabolism except L-arabinono-γ-lactonase were inducible. Assays for such enzymes in snake bean bacteroids indicated that L-arabinose did not reach the bacteroids in large quantities. The Tn5-induced mutant MNF3045 of R. leguminosarum was unable to grow on L-arabinose and accumulated L-arabinono-γ-lactone and L-arabinonate. Product accumulation and enzyme assays suggested that this mutant was defective in L-arabinonate dehydratase. It nodulated peas and the nodules fixed N2, indicating that the supply of L-arabinose is not essential for bacteroid function. Another Tn5-induced mutant of R. leguminosarum, MNF3041, lacked ribokinase and was unable to grow on D-ribose; this mutant was also able to nodulate peas and fix N2.
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Properties of Double Mutants of Rhizobium leguminosarum Which Are Defective in the Utilization of Dicarboxylic Acids and Sugars
More LessSUMMARY: Rhizobium leguminosarum MNF3201 is a mutant defective in C4 dicarboxylic acid transport (dct). which forms ineffective nodules on pea. The Tn5-induced mutants MNF3041 (ribokinase-negative), MNF3045 (arabinonate dehydratase-negative), MNF3064 (defective in the “Entner-Doudoroff enzymes”) and MNF3070 (pyruvate carboxylase-negative) nodulate peas and fix N2. The carbohydrate-utilization mutations of these strains were each transduced into MNF3201 using phage RL38. The double mutants were defective in the utilization of both C4 dicarboxylic acids and the respective carbohydrates. All the double mutants formed nodules on peas, although these were still ineffective. Thus it appears that R. leguminosarum can use carbon sources other than dicarboxylic acids or common classes of sugars to fuel the nodulation process.
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Propargylglycine as a Fungal Inhibitor: Effect on Sulphur Amino Acid Metabolism
More LessSUMMARY: The effect of propargylglycine (PPG) on four fungal species was studied. γ-Cystathionase was the major target of the antibiotic, as in mammalian but not in bacterial cells. Highly PPG-sensitive γ-cystathionase was found in Aspergillus nidulans and Neurospora crassa, less sensitive in Saccharomyces cerevisiae and insensitive in Cephalosporium acremonium. The effects of γ-cystathionase inhibition on growth depend on the role this enzyme plays in cysteine synthesis. Inhibition by PPG was reversed by cysteine, and also by other amino acids at high concentrations. The results of transport assays of wild-type strains and PPG-resistant mutants indicated that propargylglycine was taken up by amino acid permeases in A. nidulans and S. cerevisiae.
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Influence of L-Sorbose on Growth and Enzyme Synthesis of Trichoderma reesei C-5
More LessSUMMARY: The effects of L-sorbose on the growth and biosynthesis of cellulases and other polysaccharide-degrading enzymes of Trichoderma reesei C-5 were studied. The specific growth rate and yield of this strain in batch culture were reduced by 23% and 46% respectively on addition of 1% (w/v) sorbose to Vogel's medium containing 1% (w/v) glucose. The specific consumption rate of both sorbose and glucose decreased in the presence of the other sugar at 1% (w/v) concentration. The addition of sorbose (1-5%) to cultures grown in 1% glucose resulted in enhanced activities of all cellulase enzymes, and particularly endoglucanase activity, which increased sevenfold in the presence of 5% sorbose. There was no significant effect on the activities of β-glucosidase, acid phosphatase and amylase. While the increased enzyme activities seemed to be correlated with a decreased rate of glucose consumption, a direct effect on some extracellular enzymes could not be ruled out.
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Thioglycerol Inhibition of Growth and Aflatoxin Production in Aspergillus parasiticus
More LessSUMMARY: The effects of thioglycerol on growth and aflatoxin production by Aspergillus parasiticus were studied using conidia-initiated cultures and pregrown mycelia. Thioglycerol inhibited both growth and aflatoxin synthesis, with toxin formation being affected to a greater extent. This inhibitory activity could not be overcome by addition of methionine or zinc sulphate. Accompanying respirometric studies suggested that the primary mode of action of thioglycerol involves an inhibition of oxygen utilization.
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Sexual Activation of Carotenogenesis in Phycomyces blakesleeanus
More LessSUMMARY: Sexual stimulation increases the β-carotene content of the fungus Phycomyces blakesleeanus. The same effect is observed in single cultures exposed to natural and synthetic trisporates and in intersexual heterokaryons. Synthetic racemic (9Z)-methyl trisporate B, a trisporate precursor made by cultures of the (+) mating type, stimulates carotenogenesis only in (-) cultures. Synthetic racemic (9Z)-methyl trisporate C is less effective and the corresponding all-(E) isomers and other related compounds are inactive. Sexual stimulation of carotenogenesis is additive with the stimulations induced by light, retinol, dimethyl phthalate, and mutation of the gene carS.
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Induction of Macrocyst Germination in the Cellular Slime Mould Dictyostelium mucoroides
More LessSUMMARY: Despite the usefulness of the macrocyst cycle for genetic analysis of slime mould cells, a great difficulty has been the extremely poor germination of the macrocysts. We describe culture conditions for reliable, high-frequency macrocyst germination, using Dictyostelium mucoroides 7 (Dm7) and a mutant MF1 derived from it. Irradiation with relatively short-wavelength light (blue-near-UV) was particularly important for the induction of germination, and only a short period of irradiation was required to obtain a high percentage of germination. There was a critical temperature between 19 and 22 °C for efficient induction of germination; a relatively high pH, around 8.0, also favoured germination. Germination competence increased with the age of the macrocysts.
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Sinapic Acid Degradation by the Yeast Rhodotorula glutinis
More LessSUMMARY: The degradation of sinapic acid, a monomer of hardwood lignins, by the yeast Rhodotorula glutinis was studied. Syringic acid, 3-O-methyl gallic acid, gallic acid and 2,6-dimethoxy-1,4-benzoquinone were identified as degradation products. Glucose was shown to be required for the demethylation of the methoxy groups on the ring. Unlike in the bacterium Pseudomonas putida, ring cleavage seemed to occur via gallic acid and not via methyl gallic acid. Cell-free oxidative decarboxylase (hydroxylase) activity was detected; this enzyme might be ultimately responsible for the formation of dimethoxyquinone.
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Formation of an Extracellular Laccase by a Schizophyllum commune Dikaryon
More LessSUMMARY: A dikaryon of the basidiomycete Schizophyllum commune growing in surface culture at 30 °C in the dark produced extracellular laccase (EC 1.10.3.2). Little extracellular laccase was formed in the light or at 24 °C. The co-isogenic monokaryons from which the dikaryon was generated generally failed to produce laccase. The activity in the medium of the dikaryon accumulated until the glucose was consumed and then declined steadily. At its peak level of activity the enzyme accounted for about 3% of the extracellular protein. The enzyme was purified by DEAE-Sephacel chromatography. Substrate specificity and inhibitor studies showed the enzyme to be a typical fungal laccase. Electrophoresis of a purified enzyme preparation showed one major band accounting for 98% of the laccase proteins, and two minor bands with laccase activity. The major laccase protein was used to raise specific antibodies. After denaturation of the major laccase two immuno-reactive protein forms of M r 64 x 103 and 62 x 103 were produced, the former being convertible into the latter form. The intracellular extract contained one immuno-reactive protein of M r 72 x 103. The presence of laccase protein in the medium of various cultures was detected using Western blots. Accumulation of extracellular laccase protein only occurred in the dikaryon at 30 °C in the dark while the subsequent decrease in activity was not accompanied by a decrease in laccase protein.
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Evidence for Identical Dichloromethane Dehalogenases in Different Methylotrophic Bacteria
More LessSUMMARY: The dichloromethane dehalogenases of four facultative methylotrophs that mineralize dichloromethane were compared. Similar levels of specific activity and a similar subunit M r of the dehalogenases were observed in crude extracts of the four strains. Immunodiffusion analysis and quantitative immunoprecipitation indicated that the dehalogenases closely resemble each other. This was confirmed when the N-terminal amino acid sequences of the four dichloromethane dehalogenases were found to be identical. Some variation was observed with respect to the specificity of the inducers for dichloromethane dehalogenase. In the two Hyphomicrobium strains examined 1,1-dichloroethane and 1,2-dichloroethane were effective gratuitous inducers whereas these compounds exhibited only a marginal inducing effect in the two Pseudomonas strains. The results of this comparative study are compatible with the view that dichloromethane dehalogenase represents a recently evolved enzyme which was horizontally distributed by gene transfer.
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- Systematics
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A Cooperative Taxonomic Study of Mycobacteria Isolated from Armadillos Infected with Mycobacterium leprae
SUMMARY: Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.
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Pseudomonas fluorescens Biovar V: Its Resolution into Distinct Component Groups and the Relationship of These Groups to Other P. fluorescens Biovars, to P. putida, and to Psychrotrophic Pseudomonads Associated with Food Spoilage
More LessSUMMARY: A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.
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Characterization of Flavobacterium Species by Analysis of Volatile Fatty Acid Production
More LessSUMMARY: Seventy-four Flavobacterium strains were characterized by gas-liquid chromatographic analysis of volatile fatty acids produced in the culture medium. Principal components analysis permitted the graphic representation of the relative positions of the different strains, and aggregation according to the variance enabled a hierarchical classification to be established. The study revealed three subgroups each for F. meningosepticum and F. odoratum. Our F. breve, Flavobacterium sp. group IIb and F. multivorum strains appeared to be homogeneous. These results tallied with those of previous studies on DNA base composition and reassociation, electrophoretic protein profiles and cellular fatty acid composition.
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Isolation of a New Free-living Bacterium Containing R-bodies
More LessSUMMARY: Strain EPS-5028 is a free-living bacterium which contains R-bodies. By its morphology, biochemical characteristics and G + C content, strain EPS-5028 differs from the other known free-living R-body-containing bacteria, Pseudomonas avenae and Pseudomonas taeniospiralis. Phage and particles resembling phage heads were observed in cells induced with UV irradiation.
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Inhibition of Restriction in Streptomyces clavuligerus by Heat Treatment
More LessSUMMARY: Inefficient transformation of Streptomyces clavuligerus protoplasts by DNA from the plasmid pIJ702, isolated from S. lividans, was attributed to restriction in view of the observation that efficient transformation was observed using modified pIJ702 (isolated from S. clavuligerus). The restriction system could be partially inhibited by treating protoplasts at 45 °C prior to transformation. This treatment increased the transformation frequencies of pIJ702 DNA by 100-fold and was used to introduce other plasmids into S. clavuligerus.
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Evidence for the Role of a Siderophore in Promoting Vibrio anguillarum Infections
More LessSUMMARY: Vibrio anguillarum strain 775 harbouring the virulence plasmid pJM1 produces a plasmid-mediated siderophore that can crossfeed siderophore-deficient, receptor-proficient mutants of V. anguillarum in vitro. Experimental infections of salmonid fishes with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-proficient mutant resulted in recovery of both the wild-type and the mutant strain, while in infections with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-deficient mutant only the wild-type strain could be recovered. These results suggest that the V. anguillarum plasmid-mediated siderophore is produced in vivo in a diffusible form and that it is an important factor of virulence.
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