- Volume 131, Issue 8, 1985
Volume 131, Issue 8, 1985
- Sgm Special Lecture
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Little Fleas and Lesser Fleas
More LessSummary: Ruder heads stand amazed at these prodigeous pieces of nature, whales, elephants, dromedaries, and camels; these, I confess, are the colossus and majestick pieces of her hand; but in these narrow engines there is more curious mathematicks; and the civility of these little citizens more easily sets forth the wisdom of their Maker.
Sir Thomas Browne, 1642
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- Biochemistry
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An Inducible Xylanase of the Mushroom Termitomyces clypeatus Differing from the Xylanase/Amylase Produced in Dextrin Medium
More LessSummary: The mushroom Termitomyces clypeatus produces a single endoxylanase (1,4-β-D-xylan xylano-hydrolase, EC 3.2.1.8) in the presence of either dextrin or xylan as sole source of carbon. The enzymes produced in the two conditions are different. The enzyme induced by xylan has been purified 67-fold from the culture filtrate of T. clypeatus. The enzyme preparation gave a single protein band on SDS-PAGE, corresponding to a molecular weight of about 24000. The enzyme has an isoelectric point at pH 4·0 and acts on arabinoxylan and arabinogalactan, but not amylopectin or galactomannan. It shows maximum activity on xylan (1,4-β-linked D-xylopyranose units) at pH 3·5 and 55°C and is fairly stable up to 60°C. The K m for xylan is 4 mg ml−1. Hg2+, Fe2+ and Ag+ are the most potent inhibitors of the enzyme. The pH optimum and molecular weight of this inducible xylanase differ from those of the enzyme produced by the same organism grown in dextrin medium.
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One-dimensional Peptide Mapping of the Subunits of Pertussis Toxin
More LessSummary: Pertussis toxin (pertussigen) purified from the cytoplasmic fraction of Bordetella pertussis strain 18334, phase 1, consisted of five subunits which included an additional subunit (S1a) not previously reported. Subunits S1, S1a and S2 showed extensive structural homology when analysed by one-dimensional peptide mapping, indicating that the latter two were probably derived from proteolytic cleavage of the largest subunit, S1. Subunits S3 and S4,5 generated only a limited number of peptides following chemical and enzymic degradation, but these subunits differed structurally from each other and from those showing structural homology.
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Glutamine Synthetase of Streptomyces cattleya: Purification and Regulation of Synthesis
More LessSummary: Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.
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Purification and Molecular and Catalytic Properties of Bromoperoxidase from Streptomyces phaeochromogenes
More LessSummary: A bromoperoxidase has been isolated and purified from the chloramphenicol-producing actinomycete Streptomyces phaeochromogenes. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis. The prosthetic group of the bromoperoxidase was ferriprotoporphyrin IX. Based on gel filtration results the molecular weight of the enzyme was 147000 ± 3000. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a single band having the mobility of a 72500 molecular weight species. Therefore, in solution at neutral pH, the bromoperoxidase behaved as a dimer. The isoelectric point was 4·0. The spectral properties of the native and reduced enzyme are reported. The homogeneous enzyme also had peroxidase and catalase activity.
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The Purification and Properties of Cellobiose Dehydrogenase from Sclerotium rolfsii and its Role in Cellulolysis
More LessSummary: An extracellular cellobiose dehydrogenase has been purified from the culture filtrates of Sclerotium rolfsii. The purified enzyme is homogeneous as determined by disc gel electrophoresis, with and without SDS, and by analytical isoelectric focusing in polyacrylamide gel. The enzyme is a single-subunit glycoprotein containing 8·9% total carbohydrate; its M r is 63000-64500, and its isoelectric point 5·18. The enzyme oxidized cellobiose, other cellodextrins and lactose whereas other disaccharides tested were not utilized as substrates. The rate of cellodextrin oxidation decreased and the K m increased with increasing degree of polymerization of the substrate. Cytochrome c was reduced though at a considerably lower rate than 2,6-dichlorophenolindophenol. The natural electron acceptor for the enzyme has not been identified.
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Purification and Properties of LIQ 4, an Antibacterial Substance Produced by Streptococcus faecalis var. liquefaciens K 4
More LessSummary: An antibacterial substance (LIQ 4) produced by Streptococcus faecalis var. liquefaciens K 4 was isolated in extracellular and cell associated form. It markedly inhibited the growth of Gram-positive bacteria, whereas only a few Gram-negative bacteria were susceptible. LIQ 4 was purified by hydrophobic chromatography (Servachrome XAD-2; octyl-Sepharose CL-4B; Sephadex LH 60) and Sephacryl S-200. TLC yielded a fluorescent spot as the active component and several inactive ninhydrin-positive substances. These contaminating peptides strongly adsorbed to LIQ 4 and could only be removed by repeated reversed phase HPLC. Furthermore, HPLC separated LIQ 4 into seven closely related substances. All showed strong fluorescence under UV light, stained yellow with ninhydrin, and contained aspartate and lysine after acid hydrolysis. The molecular weight was estimated by Amicon ultrafiltration to be less than 2000. LIQ 4 was stable at 80°C (30 min) and pH 2, but considerably inactivated above pH 8. It was apparently affected by proteolytic enzymes, but the activity could be fully restored upon heating.
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One or Two Low Affinity Penicillin-binding Proteins May Be Responsible for the Range of Susceptibility of Enterococcus faecium to Benzylpenicillin
More LessSummary: Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 μ;g ml−1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0·031-0·5 μ;g ml−1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 μ;g ml−1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0·062-128 μ;g ml−1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.
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Composition of Lipopolysaccharide from Strains of Pseudomonas syringae pv. morsprunorum of Differing Host Specificity and Virulence
More LessSummary: Lipopolysaccharide (LPS) from strains of Pseudomonas syringae pv. morsprunorum isolated from plum and cherry contained a uniform core region, which, in all cases, was composed of 2-keto-3-deoxyoctonic acid, heptose, glucosamine, galactosamine, rhamnose, glucose, alanine, ethanol-amine and phosphate. Lipid A contained glucosamine, phosphate and the fatty acids 3-OH 10:0, 12:0, 2-OH 12:0, and 3-OH 12:0. Sidechains of LPS from two virulent plum isolates were composed of glucose and rhamnose, and were susceptible to hydrolysis by rhamnanase from the typing phage A7, which uses LPS as its binding site. Phage A7-resistant mutants of the virulent cherry isolate C28 contained rough LPS, did not adsorb A7, and displayed reduced virulence towards cherry. An avirulent, phage A7-resistant mutant of a plum isolate contained smooth LPS with sidechains of a high glucose content that were resistant to phage hydrolysis. Loss or alteration of sidechains therefore correlated with loss of virulence.
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Effects of Nitrate, Nitrite and Diphenylamine on the Photosynthetic Apparatus of Rhodopseudomonas sphaeroides f. sp. denitrificans
More LessSummary: We have compared the effect of diphenylamine (DPA) on the pigment composition of Rhodopseudomonas sphaeroides f. sp. denitrificans grown in photoheterotrophic culture with the previously reported effect of nitrate. It was demonstrated that the effect of nitrate is due to the nitrite which is produced during denitrification. Both nitrite and DPA caused a decrease in the synthesis of spheroidene, and the accumulation of more-reduced precursors not normally seen. Nitrate (effectively nitrite) caused a decrease in the amount of bacteriochlorophyll, and the reaction centre from denitrifying cells did not contain the 28 kDal polypeptide (RC-H) subunit. These effects did not occur over a range of DPA additions (4 to 8 μ;g ml−1) to cells growing in the absence of nitrate. Denitrifying cells also had 40-50% lower activity of δ-aminolaevulinic acid synthase than those grown with or without DPA. Both nitrite and DPA treatments resulted in the loss of the B870 light-harvesting complex because of a failure to synthesize its 12 kDal polypeptide subunit.
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Studies of the 2':3'-Cyclic Nucleotide Phosphodiesterase of Haemophilus influenzae
More LessSummary: The 2':3'-cyclic nucleotide phosphodiesterase:3'-nucleotidase of Haemophilus influenzae was purified from a periplasmic preparation by affinity chromatographic techniques. The enzyme-catalysed hydrolysis of 2': 3'-cyclic AMP to adenosine without accumulation of the intermediate substrate 3'-AMP was demonstrated by high performance liquid chromatography. Competitive inhibition of the enzyme by a variety of nucleosides and mononucleotides indicated the presence of either purine or pyrimidine bases to be essential for selective interactions with the enzyme, and confirmed the need for a 3'-position phosphate for the functioning of mononucleotides as substrates for the enzyme. The enzyme had a molecular weight of 79000, was stable at low temperatures and was thermally denatured at temperatures above 50°C.
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Isolation and Characterization of the Pigment Esters of Xanthomonas juglandis (campestris)
More LessSummary: Separation of the pigment esters of Xanthomonas juglandis (campestris) on silica gel columns yielded four bands. Two of the bands, esters 1 and 2, made up over 95% of the mixture. Both esters were phospholipids, with ester 1 containing the alcohol moieties glycerol and sorbitol, and ester 2 only glycerol. Both appeared to be mixtures containing different xanthomonadin pigment molecules but the results suggested that ester 1 consisted of one pigment molecule esterified to glycerophosphoryl sorbitol, whereas ester 2 consisted of 2 pigment molecules esterified to glycerophosphate.
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An Exo-D-galacturonanase of Butyrivibrio fibrisolvens from the Bovine Rumen
More LessSummary: An intracellular pectinolytic enzyme was isolated from a cell extract of Butyrivibrio fibrisolvens and purified. The optimum pH for enzyme activity was 5·6. The enzyme preferentially degraded de-esterified substrates by hydrolysis of monosaccharide units from the non-reducing end; the only product of degradation was D-galacturonic acid. Values of K m and V max for oligo- and polygalacturonates indicated that the best substrate was digalacturonic acid; oligogalact-uronates containing either a saturated or a Δ4,5-unsaturated non-reducing end were both degraded. The enzyme was classified as an exo-D-galacturonanase [poly(1,4-α-D-galacturonide) galacturonohydrolase (EC 3.2.1.67)].
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- Development And Structure
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Purification and Characterization of Flagella from Roseburia cecicola, an Obligately Anaerobic Bacterium
More LessSummary: Flagella from Roseburia cecicola, an obligately anaerobic bacterium originally isolated from murine caecal mucosa, were purified by mechanical shearing followed by differential centrifugation. Purity of the flagellar preparation was determined by polyacrylamide gel electrophoresis, electron microscopy and chemical analysis. The flagella were composed of a single protein subunit (flagellin) with an estimated molecular weight of 42000. The amino acid composition of the flagellin was similar to that of some facultatively anaerobic and aerobic bacteria.
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- Genetics And Molecular Biology
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Identification of a New Locus in the Escherichia coli Cotransduction Gap that Represents a New Genetic Component of the L-Asparagine Utilization System
More LessSummary: A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutation (asu) produces two distinct phenotypic effects. (1) Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine. (2) Mutant strains utilize L-asparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains. The mutation maps at 32·4 min on the E. coli chromosome, within the E. coli cotransduction gap. Mutant strains produce normal amounts of thermo-stable L-asparaginase I activity. The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.
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Transduction Complicates the Detection of Conjugative Ability in Lysogenic Salmonella Strains
More LessSummary: When lysogenic salmonella strains were examined for conjugative ability in tri-parental crosses, false positive results were sometimes obtained because phage carried by the lysogenic strains multiplied on the intermediate salmonella recipient strain and then transduced its streptomycin/sulphonamide resistance plasmid to the final salmonella recipient strain. Back transfer of the plasmid to the lysogenic strains was also detected.
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Resistance Plasmids of Aeromonads
More LessSummary: The resistance plasmids of isolates of Aeromonas hydrophila and A. salmonicida from Japan, France, the UK and Ireland have been characterized. Three classes of plasmids transmissible to Escherichia coli were found: plasmids of incompatibility group C, plasmids of incompatibility group U, and plasmids incapable of stable inheritance in E. coli, not belonging to any defined incompatibility group.
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Identification of Restriction Fragments from Two Cryptic Clostridium butyricum Plasmids That Promote the Establishment of a Replication-defective Plasmid in Bacillus subtilis
More LessSummary: Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6·05 kbp) and pCB102 (7·8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3·3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+and Rec−strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3·3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2·0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.
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Genetic Analysis of Candida albicans Morphological Mutants
More LessSummary: In contrast to some other strains, Candida albicans 1001 gave rise, upon UV irradiation, to mutants displaying a ‘rough colony’ morphology associated with a permanent alteration in morphogenesis which determined growth of the cells mostly as pseudohyphae. One of these mutants, C. albicans 1001FR, could form sectored (rough/smooth) colonies spontaneously, and with increasing frequency treatment with mild UV doses (32-64 μ;J mm−2). Rough sectors corresponded to stable ‘rough-filamentous’ strains which never segregated smooth strains. On the other hand, smooth sectors consisted mainly of yeast cells which could occasionally revert to a rough-filamentous phenotype. We suggest that C. albicans 1001 is heterozygous for some gene involved in the control of morphogenesis, and that the described mutants should be of help in the characterization of the genetic control of dimorphism in C. albicans.
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- Immunology
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Immunomodulation by Myxospores of Myxococcus xanthus
More LessSummary: Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 × 108 myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 × los myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 × 108 myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.
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