- Volume 117, Issue 2, 1980
Volume 117, Issue 2, 1980
- Biochemistry
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N-Methylglutamate Dehydrogenase, a Flavohaemoprotein Purified from a New Pink Trimethylamine-utilizing Bacterium
More LessA new strain of pink facultative methylotroph, bacterium AT2, capable of growth on methylamine, trimethylamine, methanol, formate and a range of non-C1 substrates has been isolated. On the basis of the enzyme activities in cell-free extracts, the organism appears to have the same pathway for trimethylamine oxidation as Pseudomonas aminovorans, i.e. via trimethylamine N-oxide and N-methylglutamate. N-Methylglutamate dehydrogenase in this organism was a ‘soluble’ enzyme (i.e. was not sedimented at 100000 g in 60 min) and reacted with the electron acceptors phenazine methosulphate, 2,6-dichlorophenolindophenol, Wurster’s blue and the radical cation of 2,2′-azinodi-[3-ethylbenzthiazoline 6-sulphonate]. It was not active with NAD, ferricyanide or cytochrome c. The enzyme was purified to apparent homogeneity (as assessed by polyacrylamide gel electrophoresis) and was shown to contain flavin and cytochrome c, both of which could be reduced by N-methylglutamate. The flavin prosthetic group could be liberated by boiling and was probably FAD. The true K m of the enzyme for N-methylglutamate was 0·33 mm and for 2,6-dichlorophenolindophenol, 0·20 mm.
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Partial Purification and Characterization of a Dye-linked Formaldehyde Dehydrogenase from Hyphomierobium X
More LessAll organisms surveyed among a range of methylotrophic bacteria growing on both C1 and multi-carbon substrates contained a dye-linked formaldehyde dehydrogenase. The activity of this enzyme was very low in bacteria grown on C1 compounds when compared to the activities of other C1-specific enzymes and was not induced during growth on C1 compounds. The enzyme was partially purified (11-fold) from methanol- and ethanol-grown Hyphomicrobium X. Though not homogeneous, it did not contain methanol dehydrogenase, formate dehydrogenase or cytochrome c. Methylphenazonium methyl-sulphate (PMS), cytochrome c, Wurster’s Blue, dichlorophenol-indophenol (DCPIP) and methylphenazonium ethosulphate (PES) could act as the primary electron acceptors in the assay system with apparent K m values for the first three substances of 0·69, 128 and 3·07 μm, respectively. The activity of the partially purified enzyme could be maintained over 3 months at -20° in the presence of ethanol (10%, v/v). The molecular weight of the enzyme was 83500 ± 2500. It was active from pH 6·5 to 9·0 with maximum activity at pH 7·2 using cytochrome c as the electron acceptor and at pH 7·6 when PMS and DCPIP were used. Several different aldehydes could act as substrates; the apparent K m values for formaldehyde, acetaldehyde, propionaldehyde and heptaldehyde were 2860, 270, 13 and 2·5μm, respectively. Methanol and ethanol were not substrates. The dye-linked formaldehyde dehydrogenase is probably a general aldehyde dehydrogenase not directly involved in the dissimilation of C1 compounds.
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The Mitochondrial Cytochromes of an Unusual Budding Yeast, Sterigmatomyces halophilus: Spectral Characterization Exploiting Fourth-order Finite Difference Analysis
More LessLow-temperature difference spectra of gradient-purified mitochondria from Sterigmatomyces halophilus revealed the presence of a-, b- and c-type cytochromes, spectrally similar to those of other yeasts. Fourth-order finite difference analysis resolved the broad a-band of b-and c-type cytochromes into eight peaks. Absorption maxima at about 539, 543·5 and 547·5 nm were attributed to one, or perhaps two, cytochrome(s) c that are loosely bound to the mitochondrial membrane. Cytochrome c 1 was identified at 5505 to 5515 nm. Maxima at about 554, 556, 559 and 562 nm were attributed to three or four distinct b-type cytochromes on the basis of their differential reduction by NADH, dithionite, or ascorbate plus N, N,-N′, N′,-tetramethyl-p-phenylenediamine in the absence or presence of antimycin. Difference spectra in the presence of CO or cyanide indicated the presence of cytochromes a (600 nm) and a 3 (608 nm). Finite difference analysis of the cytochromes c oxidase peak centred at 600 nm revealed two components; the major component at 600 nm was identified as cytochrome a and the minor component at 605 nm as cytochrome a 3 . A further CO-binding haemoprotein was tentatively identified as cytochrome o.
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Production, Purification and Properties of Extracellular Laccase of Agaricus bisporus
More LessExtracellular laccase (EC 1.14.18.1) of the cultivated mushroom Agaricus bisporus was produced constitutively in defined or complex media. No enzyme induction was found after treatment with cycloheximide or with other potential inducers such as toluidine or xylidine. The enzyme was purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography, gel filtration and affinity chromatography. It eluted as a single peak from ion-exchange, gel filtration and affinity columns and sedimented as a single band on centrifugation. It showed four enzymically active bands on electrophoresis and a diffuse band on isoelectric focusing. Its molecular weight was estimated to be about 100000 and the enzyme contained 15% carbohydrate and two atoms of copper per molecule. The substrate specificity was similar to that of other fungal laccases. Antiserum prepared against the purified enzyme gave one major precipitin band.
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Inactivation of Extracellular Laccase During Fruiting of Agaricus bisporus
More LessThe decline in activity of extracellular laccase of Agaricus bisporus which occurs during fruit body development was shown to be due to both enzyme inactivation and proteolysis. Extracts of high activity, from cultures during mycelial growth, and of low activity, from cultures during fruiting, remained unchanged on prolonged incubation and did not cross-activate or inhibit on mixing. No activity changes occurred after dialysis, gel filtration or ultrafiltration of both forms. Molecular weights, pH optima, substrate specificities and immunological properties of the two forms were identical. Differences were found in the electrophoretic profiles of the two forms of enzyme. Loss of enzyme activity was shown to be greater than loss of enzyme protein by separate immunological and electrophoretic methods.
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Regulation of Cellular Metabolism During Synthesis and Expression of the Luminous System in Beneckea and Photobacterium
More LessParameters of cellular energetics (ATP pools, adenylate energy charge, GTP pools and oxygen consumption) as well as bioluminescence and growth have been examined in batch cultures of four different species of luminous bacteria. The findings indicate that all of these energetic parameters remain constant throughout the growth cycle while bioluminescence is induced and increases many fold. These observations hold true for very bright strains during their dim and bright phases of growth, as well as for a luminescence-conditional strain under bright or dark conditions. The percentage of the total oxygen consumption that was due to bioluminescence was shown to vary by as much as a factor of 103 during growth. For very bright cells, the oxygen consumption experiments suggest both that the energetic requirements of the bioluminescent system are significant, and that the quantum efficiency (Q o2 ) of the luciferase in vivo is quite high, possibly approaching 10. Similar considerations based on ATP pool size and energetic estimates of luminescence indicate that the luminous system in bright cells represents a small but possibly significant energy drain. Finally, two methodological features are discussed. First, it was shown that extracts of cells that have been grown in media devoid of inorganic phosphate contain a heat-stable ATPase activity, which can lead to falsely low ATP values. Second, it was shown that the interfering effect of GTP on the assay of ATP could be completely overcome by the addition of GDP to the assay mixture.
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The Uptake and Metabolism of Bacteria, Amino Acids, Glucose and Starch by the Spined and Spineless Forms of the Rumen Ciliate Entodinium caudatum
More LessSpined and spineless forms of Entodinium caudatum were obtained by growth in vivo in the presence and absence, respectively, of Entodinium bursa. Washed suspensions of both forms engulfed all the bacteria tested although the spined form took them up 1·3 to 1·9 times more rapidly per unit volume of protozoon than did the spineless form. Butyrivibrio fibrisolvens and Selenomonas ruminantium were rapidly digested by the spined form after engulfment. Free amino acids were taken up on average 3·1 times and glucose approximately 60 times faster per unit volume of protozoon by the spined form. Limited amounts of protein were synthesized by the spined form from glucose and starch but engulfed bacteria and, to a lesser extent, free amino acids were probably the prinicpal sources of protein for growth of both forms.
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Regulation of the Plasmid-specified Naphthalene Catabolic Pathway of Pseudomonas putida
More LessThe regulation of the catabolic pathway for the degradation of naphthalene specified by the plasmids NAH, pND140 and pND160 was studied using plasmid-borne regulatory mutants in a Pseudomonas putida host strain. Growth of strains harbouring the parent plasmids in the presence of salicylate resulted in induction of selected enzymes involved in the conversion of naphthalene to catechol (naphthalene oxygenase, salicylaldehyde dehydrogenase and salicylate hydroxylase) and enzymes of the meta-cleavage pathway (catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semi-aldehyde dehydrogenase). Partial induction was also observed for all the NAH-encoded enzymes assayed when using m-toluate as the inducing compound, over a genetic block. Mutants were obtained for each plasmid where the three enzymes of the meta-cleavage pathway were produced constitutively suggesting that the enzymes of the meta-cleavage pathway belong to one operon. In these mutants, enzymes involved in the conversion of naphthalene to catechol were not produced constitutively but remained inducible during growth on salicylate indicating that these enzymes belong to a separate operon or operons.
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- Ecology
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Some Differences in the Microbiology of Profundal and Littoral Lake Sediments
More LessThe microbial populations of the littoral (shallow water) and profundal (deep water) surface sediments of Blelham Tarn and the South Basin of Windermere were examined. Microbial numbers (direct counts), biomass (ATP) and activity (electron transport system activity, CO2 evolution and [14C]glucose mineralization) were consistently higher in the profundal zone of both lakes, the difference being greater and average values higher in the more productive Blelham Tarn. The interstitial water of the profundal sediments contained higher concentrations of available substrates (carbohydrate, protein and amino acids). There were marked differences in the particle size distribution of the sediments with a greater proportion of small particles (< 45 μm in size) in the profundal samples. The smaller particles from both sites were colonized by a larger number of bacteria and contained higher electron transport system activity per gram dry weight. There was a greater diversity of benthic animals in the littoral zone of both lakes but larger numbers of ciliated protozoa were observed in the profundal sediments.
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Distribution and Abundance of Bacteria in the Gut of a Soil-feeding Termite Procubitermes aburiensis (Termitidae, Termitinae)
More LessThe alimentary canal of a representative species of soil-feeding termite was examined for associations with bacteria. Enumerations made in the principal regions of the intestine by direct observation and expressed for comparative purposes as total microbial standing crop showed a net three- to fourfold increase between the foregut (crop) and rectum. Filamentous organisms, putatively actinomycetes, contributed significantly to the flora in most regions of the gut and were more abundant, relative to non-filamentous forms, than in freshly ingested soil. Transmission and scanning electron microscopy of the gut wall showed that the actinomycetes formed novel associations with the host in the mesenteron, mixed segment and colon. Non-filamentous organisms, chiefly rods, colonized the walls of the first proctodaeal segment and the colon, in addition to filaments, and were present in large numbers in the contents of the third proctodaeal segment.
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- Genetics And Molecular Biology
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Characterization of Bacteriophage SPP1 Transducing Particles
More LessBacillus subtilis lysates produced by virulent bacteriophage SPP1 retained their transducing ability upon purification from contaminating PBSX particles. The buoyant density in CsCl of the transducing activity was indistinguishable from that of the SPP1 plaque-forming units and the sedimentation behaviour in sucrose gradients of purified transducing particles was the same as that of SPP1 phage particles. Further, high concentrations of anti-SPP1 serum inactivated transducing particles and SPP1 plaque-forming units at the same rate. The transduction process was resistant to DNAase treatment, but was enhanced by temperatures that did not allow transformation. It was concluded that particles of the size, shape, density and serum-sensitivity characteristic of SPP1, but carrying bacterial DNA, are vectors in a true transduction process. Cell survival upon SPP1 infection is discussed.
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Amino-sugar Transport Systems of Escherichia coli K12
More LessGlucosamine, mannose and 2-deoxyglucose enter Escherichia coli by the phosphotransferase system coded for by the gene ptsM. The glucosamine- and mannose-negative, deoxyglucose-resistant phenotype of ptsM mutants can be suppressed by a mutation mapping near ptsG that allows constitutive expression of the glucose phosphotransferase coded for by the gene ptsG.
N-Acetylglucosamine enters E. coli by two distinct phosphotransferase systems ( White, 1970 ). One of these is the PtsM system, the other is coded for by a gene which maps near the nag A, B genes at about min 15 on the E. coli chromosome. We propose that this gene be designated ptsN. Strains with either of these components of the phosphotransferase system will utilize N-acetylglucosamine as sole carbon source.
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Two Genes Affecting Glucarate Utilization in Escherichia coli K12
More Lessd-Glucarate is transported into Escherichia coli K12 by an inducible system at an apparent rate of 7 to 15 nmol min−1 (mg dry mass)−1. The apparent K m for uptake is 16 m. The system is induced by growth on glucarate or glycollate. Galactarate competes with glucarate for the uptake system. A mutation (garA) was isolated in which activities of glucarate transport and glucarate dehydratase and the ability to grow on glucarate or galactarate are all impaired. The mutation maps at min 16. Another mutation of indistinguishable phenotype is probably a deletion of the genes garB and ton A at min 3·5.
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Isolation and Characterization of a Mutant of Escherichia coli K12 Synthesizing DNA Polymerase I and Endonuclease I Constitutively
More LessA mutant of Escherichia coli K12, highly resistant to ultraviolet radiation, has been isolated. Preliminary tests show that this mutant is also resistant to mitomycin C, nalidixic acid, fluorouracil and thymineless death. The mutant strain apparently repairs its damaged DNA more efficiently than wild-type E. coli K12 strains and, to do so, constitutively produces 35 times more DNA polymerase I and 12 times more endonuclease I than the wild-type strain.
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Conjugational Gene Transfer in Xanthobacter autotrophicus GZ29
More LessA low-frequency recombination system, requiring direct cell contact and permitting transfer of large chromosomal segments, has been found in the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GZ29. All partners used in plate mating experiments functioned as donors as well as recipients. Two groups of closely linked markers were found by recombination analysis. The mutations of a number of autotrophic-negative mutants were placed in one or other of these two linkage groups.
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Ribosomal RNA Genes in Kluyveromyces marxianus
More LessDNA from the yeast Kluyveromyces marxianus was studied for its heterogeneity and the multiplicity of rRNA cistrons. These cistrons banded at a slightly different density from the bulk DNA in preparative CsCl or Hg2+-Cs2SO4 equilibrium density gradients. The reassociation kinetics of the bulk DNA showed that the repetitive fraction represented a small amount of the total cellular DNA (10%) and that the single copy fraction had a complexity of 6·3×109 daltons. Approximately 2·2% of the DNA hybridized to 3H-labelled rRNA at saturation. On the basis of the above genome size, the multiplicity of the rRNA cistrons was calculated to be about 70 per haploid nucleus.
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Variant Pili Produced by Mutants of the Flac Plasmid
More LessTransfer-proficient Flac mutants with reduced abilities to plate various F-specific phages were isolated, either by selection after mutagenesis, or as revertants of Flac traA mutants. In many of the mutants pilus-related properties were altered, including physical adsorption of R17 phage, the number of pili per cell and the outgrowth/retraction equilibrium. Complementation studies showed that the mutations were in traA, suggesting that specific alterations in the amino-acid sequence of the pilin subunit protein were responsible for the altered pilus properties. Complementation between the Flac traA mutants and the derepressed plasmid R100-1 restored phage sensitivity in some cases, suggesting that the incorporation of both mutant and R100-1 subunits into the pilus structure may result in conformational changes which increase the capacity of the pilus to interact with phages.
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The Genome of Rhodomicrobium vannielii, a Polymorphic Prosthecate Bacterium
More LessThe base composition, of Rhodomicrobium vannielii DNA was found to be 62·2% GC, and the genome size was 2·1 × 109 daltons. There was no detectable difference between DNA from each of the three cell expressions examined. Reassociation kinetics indicated that no large group of repeated sequences was present, but that 5 % of the genome was composed of extremely rapidly reassociating sequences. No plasmids were detected. Electron microscopic examination showed that R. vannielii DNA contained short inverted repeat sequences on average 400 base pairs long. The possible function of these sequences is discussed.
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The Use of Translocatable Genetic Elements to Construct a Fine-structure Map of the Klebsiella pneumoniae Nitrogen Fixation (nif) Gene Cluster
More LessThe transposons Tn5, Tn7 and Tn10 and bacteriophage Mu have been used to derive insertion mutations in the Klebsiella pneumoniae nif gene cluster. A large number of deletion mutants have been derived by imprecise excision of insertion mutations and these deletions have been used to construct a fine-structure map of the nif cluster. Comparison of this genetic map with a physical map of the nif cluster derived by Reidel et al. (1979) showed a very good correlation between genetic and physical mapping methods.
A new complementation group, designated nifU, has been identified and mapped between nifN and nifS. Polarity studies on the 14 nif cistrons now identified suggests that they are organized in at least seven transcriptional units and that all the multicistronic units are transcribed in the same direction.
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- Medical Microbiology
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Preparation and Quantitative Determination of Antibodies Against Major Outer Membrane Proteins of Escherichia coli O26 K60
H. Hofstra and J. DankertAntisera against isolated outer membrane (OM) proteins I and II* of Escherichia coli 026 K60 were elicited in rabbits. Antisera obtained after intramuscular administration with Freund’s complete adjuvant showed high titres of specific antibodies. Intravenous administration of the same preparations yielded a considerable antibody response against bacterial lipopolysaccharide, a minor contaminant of the protein preparations. Antibody titres against OM proteins I and II* , lipopolysaccharide and murein-lipoprotein were determined by the enzyme-linked immunosorbent assay (ELISA) in these sera, and in antisera elicited against whole formaldehyde-fixed bacteria or isolated OM. Comparison of ELISA with single radial immunodiffusion and interfacial immunoprecipitation tests revealed that ELISA was not only the most uniformly applicable, but also the most specific and the most convenient method. In double diffusion tests no cross-reactivity between proteins I and II* was seen. Antibodies against proteins I and II* , lipopolysaccharide and lipoprotein could be specifically absorbed from the sera with the appropriate antigen preparations. Absorption experiments with intact E. coli O26 K60, Tris/EDTA-sheared bacteria and isolated OM revealed that antibodies against protein I were hardly absorbed at all probably because the antibody, evoked against denatured protein I, did not react with the protein in its native configuration. Antibodies against protein II* and lipoprotein were absorbed by intact as well as by sheared bacteria, but to a much greater extent by isolated OM, which indicates that these OM components are accessible from the outside, but that they are situated relatively deep in the OM structure.
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