- Volume 133, Issue 9, 1987
Volume 133, Issue 9, 1987
- Biochemistry
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Release of Pertussis Toxin and Its Interaction With Outer-membrane Antigens
More LessSUMMARY: The absence of subunit S3 in cell-associated pertussis toxin (PT) from a mutant of Bordetella pertussis which failed to produce cell-free toxin suggested that this subunit was involved in the release of PT into the culture medium. The addition of methylated β-cyclodextrin (MCD) to the culture medium caused a small but consistent increase in the release of lipopolysaccharide (LPS) by four wild-type strains of B. pertussis. Since previous studies have shown that MCD also enhances the levels of PT in culture supernates, it seemed probable that the increased shedding of outer-membrane vesicles (OMV) may explain the increased levels of both cell-free PT and LPS. Release of PT was inhibited in media buffered with HEPES but was unaffected in Tris/HCl buffer. This suggested that in addition to shedding of the outer membrane, increased permeability and greater destabilization of the outer membrane, as caused by Tris/HCl buffer, may be important in the release of PT. Our data do not support the idea that PT is packaged into OMV because only an insignificant proportion (0·01%) of the total cell-free PT was associated with LPS. The association of PT with small micelles derived from outer-membrane amphiphiles may be more important since the LPS content of PT purified from culture supernates (containing no large OMV) was nearly 18% by weight.
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Novel Structure, Properties and Inactivation of Glutamine Synthetase Cloned from Bacteroides fragilis
More LessSUMMARY: The cloned Bacteroides fragilis glutamine synthetase (GS) subunit produced in Escherichia coli had the same apparent M r of approximately 75000 as the GS subunit from B. fragilis cells. The B. fragilis GS enzyme had an apparent M r of approximately 490000 and it is concluded that the GS is a hexamer. The cloned GS did not appear to be regulated by adenylylation and deadenylylation and the cloned enzyme was inactivated by snake venom phosphodiesterase. The pH profiles of the cloned GS, assayed by the γ-glutamyl transferase (GGT) assay were similar for NH4 +-shocked and unshocked cell extracts and an isoactivity point was not obtained from these curves. The cloned GS was subject to feedback inhibition by amino acids but not by AMP. The GGT activity of the cloned GS in NH4 +-shocked and unshocked cell-free extracts was inhibited by Mg2+. Mn2+ stimulated the cloned GS GGT activity of NH4 +-shocked cell-free extracts. Western blotting indicated that GS production was regulated by nitrogen in B. fragilis cells but cell extracts showed no GGT activity. Cloned B. fragilis GS produced in E. coli was specifically and irreversibly inactivated by B. fragilis cell extracts.
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NADP-Specific Isocitrate Dehydrogenase of Mycobacterium phlei ATCC 354: Purification and Characterization
More LessSUMMARY: NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8·5. The K m values for isocitrate and NADP were 74 and 53 μm, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70°C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.
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The Cytochromes of Acetobacter pasteurianus NCIB 6428. Evidence of a Role for a Cytochrome a1-like Haemoprotein in Electron Transfer to Cytochrome Oxidase d
More LessSUMMARY: Cells from oxygen-limited cultures of Acetobacter pasteurianus NCIB 6428 contained cytochrome d, a 596 nm absorbance (in reduced minus oxidized difference spectra at room temperature) similar to cytochrome a 1, and b-type cytochromes, including a cytochrome o-like pigment. Oxygen-sufficient cells lacked the d and a 1-like components. Pyridine haemochrome spectra suggested the presence of haem a. Photolysis with white light at −126°C of an anoxic suspension of reduced oxygen-limited cells, into which CO had been bubbled, elicited a photodissociation spectrum that revealed the cytochrome o-like species, but not cytochromes d or a 1. When a similar suspension, but to which O2 had been added at −23°C, was photolysed at −126 to −132°C, using white light or irradiation with a He-Ne laser, an additional, intense absorbance at 648 nm (relative to the CO-liganded, reduced form) was observed, attributable to an oxygenated (Fe2+O2 or Fe3+O2 -) complex of cytochrome oxidase d. Photolysis at progressively warmer temperatures, or successive scans of a sample photolysed at −91°C, revealed (i) increasing depth of a trough (relative to the CO-liganded, reduced form) at 598 nm attributed to the a 1-like haemoprotein and (ii) cytochrome b oxidation. Laser photolysis of the cytochrome d-CO complex at −81°C in the presence of oxygen suggested that oxidation of cytochrome a 1 preceded that of cytochrome(s) b. It is proposed that, in this strain of Acetobacter, a cytochrome a 1,-like pigment mediates electron transfer from cytochrome(s) b to cytochrome oxidase d, but that cytochrome a 1 is not itself an oxidase.
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Biotransformation of 3-Methylphthalate by Micrococcus sp. Strain 12B
More LessSUMMARY: When Micrococcus strain 12B grown on o-phthalate was incubated with 3-methylphthalate, three compounds accumulated. These were shown to be 2-pyrone-3-methyl-4,6-dicarboxylic acid, 3,4-dihydroxy-6-methylphthalic acid, and 5-hydroxy-3-methylphthalic acid, all previously undescribed. A pathway for the formation of these compounds is proposed.
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Putrescine Breakdown in the Yeast Candida boidinii: Subcellular Location of Some of the Enzymes Involved and Properties of Two Acetamidoaldehyde Dehydrogenases
More LessSUMMARY: Two acetamidoaldehyde dehydrogenases were identified in Candida boidinii grown on putrescine as sole nitrogen source with glucose as carbon source. One of them, enzyme A, although present when cells were grown on ammonium or l-lysine, increased in activity when cells were grown on putrescine or spermidine. The other, enzyme B, was absent when the putrescine was replaced by l-lysine or ammonium, but was present if the nitrogen source was spermidine or acetylputrescine. Both dehydrogenases were active with NAD+ or NADP+ as electron acceptor. Apparent K m values for 3-acetamidopropionaldehyde and 4-acetamidobutyr-aldehyde were respectively 0·83 mM and 0·041 mM for enzyme A and 0·077 mM and 0·015 mM for enzyme B. Enzyme A was competitively inhibited by chloral hydrate with a K i of 0·6 mM, whiile enzyme B was unaffected. Both enzymes were slightly (20%) stimulated by 50 mM-KCl. Although both enzymes catalysed the oxidation of a range of aldehyde substrates, and are thus both general aldehyde dehydrogenases, it is suggested that acetamidoaldehyde dehydrogenase B is more probably specifically involved in putrescine degradation. Subcellular fractionation of spheroplast lysates showed that enzyme B was cytosolic, remaining unsedimented at 100000 g, while enzyme A co-sedimented with mitochondrial marker enzymes in a sucrose density gradient. It was also shown that acetamidoalkanoate deacetylase and acetylputrescine oxidase activities, two other key enzymes in the breakdown of putrescine and spermidine, were respectively cytosolic and peroxisomal in their location in the cell.
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Streptomyces albus G Produces an Antibiotic Complex Identical to Paulomycins A and B
J. MAJER and K. F. CHATERSUMMARY: An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with M r values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.
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Proteolysis of Hexokinase PII is Not the Triggering Signal of Carbon Catabolite Derepression in Saccharomyces cerevisiae
More LessSUMMARY: The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by d-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D. 308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.
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- Development And Structure
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A Method for Preparing Membrane Vesicles from Acetobacter aceti
More LessSUMMARY: Membrane vesicles were prepared from Acetobacter aceti by a method entailing growth in the presence of glycine, osmotic shock and lysozyme digestion. Energy-dependent transport of the amino acid valine could be demonstrated in the vesicles.
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- Genetics And Molecular Biology
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Studies of Transcriptional Regulation of the Bacillus subtilis Developmental Gene spoVE
More LessSUMMARY: The structural gene of the spoVE locus of Bacillus subtilis was replaced with the promoterless lacZ gene of Escherichia coli. The spoVE::lacZ gene fusion was transferred to the B. subtilis chromosome and β-galactosidase activity was measured under sporulation conditions. Expression of the hybrid gene could be detected as early as 40 min after the induction of sporulation. Transcription of the spoVE::lacZ gene was dependent on the products of two stage 0 loci, spo0H and spo0K. Mutations in the spoIIA and spoIIG loci did not impede expression of spoVE and, therefore, neither of the sigma factors coded for by these loci seems to be necessary for its transcription. Consequently, the spoVE locus does not seem to be part of the dependent sequence of operons involved in the developmental change, although its protein product is clearly needed for the completion of spore formation.
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Nucleotide Sequence of the Sporulation Operon, spoIIIE, of Bacillus subtilis
More LessSUMMARY: A fragment of Bacillus subtilis DNA 3490 bp long, capable of complementing spoIIIE mutations, was sequenced. The region of the fragment that encodes functions required for sporulation was delimited using integrational plasmids. Sequencing showed that this region contained an operon with two open reading frames together with associated ribosome-binding sites. The deduced translation products would be polypeptides of 518 and 252 amino acid residues. Several sequences resembling promoters recognized by RNA polymerase containing σ29 occur in the region preceding the larger open reading frame. Although no transcription-termination signal was identified downstream of the smaller coding region, analysis with integrational plasmids and determination of the size of spoIIIE messenger RNA suggest that the locus does not contain a third gene.
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Regulation of Stage II of Sporulation in Bacillus subtilis
More LessSUMMARY: A mutation, spo-87, in the spo0J locus of Bacillus subtilis allows appreciable transcription of spoIIA, spoIID and spoIIG and later operons, even though most of the cells are morphologically blocked at stage 0 and the incidence of heat-resistant spores is about 1 per 104 cells. This mutation therefore appears to disengage the genetic control of sporulation from the morphological changes to which it should be connected. spoIIA and spoIIG are transcribed independently of one another. However, the products of both operons are needed for the activation of spoIID which occurs later. This indicates a convergence of parallel pathways of operon expression. We have also shown that nonsense mutations in spoIIAC (which codes for a sigma factor of RNA polymerase) prevent transcription of spoIID; by contrast, missense mutations in the same gene allow transcription of spoIID.
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The Possible DNA-binding Nature of the Regulatory Proteins, Encoded by spoIID and gerE, Involved in the Sporulation of Bacillus subtilis
More LessSUMMARY: The predicted polypeptide products of two genes, spoIID and gerE, which appear to be concerned in the regulation of spore formation in Bacillus subtilis have been compared by modelling methods with known DNA-binding proteins. The results indicate that both polypeptides may have DNA-binding properties and the conclusion is drawn that this may account for their regulatory action.
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Isolation and Partial Characterization of Three Classes of Mutant in Pseudomonas syringae Pathovar pisi with Altered Behaviour towards Their Host, Pisum sativum
More LessSUMMARY: Mating with Escherichia coli strain SM10 carrying the Tn5 vector pSUP2011 was used to mutagenize Pseudomonas syringae pv. pisi strain 299A. The resulting transconjugants were each tested by stem-inoculation into several pea (Pisum sativum) cultivars. Three classes of mutant, which probably resulted from insertion of part or all of RP4-2-Tc:: Mu into the genome of strain 299A, showed reduced virulence towards one or more pea cultivars. The single class I mutant was avirulent on all pea cultivars tested and had lost the ability to induce a hypersensitive response in tobacco (Nicotiana tabacum) cv. White Burley; the single class II mutant induced a hypersensitive response on all pea cultivars and tobacco; class III mutants showed reduced virulence towards pea cv. Early Onward, while remaining fully virulent towards other normally susceptible pea cultivars, and inducing a hypersensitive response in tobacco.
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Cloning and Expression in Escherichia coli of Proteus vulgaris Genes for 16S Ribosomal RNA
More LessSUMMARY: In contrast to the established systems of plasmid-coded homologous ribosomal DNA (rDNA) cistrons in Escherichia coli little is known about the fate of heterologous rRNA. In order to study expression of foreign rDNA, rRNA cistrons from Proteus vulgaris were cloned in phage vector Charon 35, subcloned in pBR322 and transformed in E. coli. The inserts of two clones (pPM2 and pPM14) were characterized by restriction analysis and Southern hybridization. Each of them harboured a complete rrn cistron. The location of rRNA genes of clone pPM2 was also verified by R-loop analysis. The 5’ flanking region of the 16S rRNA of pPM2 was sequenced and compared to the E. coli counterparts. High-level homologies exist in the functional parts of this region, e.g. promoters, box A and RNAase III recognition site. The copy number of pPM2 and pPM14 was estimated to be 8 and 10, respectively. Clones showed a markedly reduced growth rate (generation time about 57 to 70 min) as compared to the non-transformed cells (generation time 40 min). rDNA cistrons of P. vulgaris were properly expressed and the transcripts are processed as demonstrated by the presence of 16S rRNA from P. vulgaris in both ribosomes and 30S ribosomal subunits isolated from the transformed E. coli cells. The fraction of heterologous rRNA in ribosomes was about 25%.
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An Unmodified Form of the ColE2 Lysis Protein, an Envelope Lipoprotein, Retains Reduced Ability to Promote Colicin E2 Release and Lysis of Producing Cells
More LessSUMMARY: Site-directed mutagenesis was used to replace the codon for the N-terminal cysteine residue of pColE2-P9-encoded mature lysis protein (CelB) by an arginine codon. In contrast to the wild-type CelB protein, the product of the mutated gene, which has an altered signal peptidase cleavage site, was neither processed nor acylated. However, the mutant protein retained sufficient residual activity to cause partial, Mg2+-suppressible lysis and could activate envelope phospholipase Al-A2 and promote colicin release, albeit with reduced efficiency compared to the wild-type protein. We propose that the uncleaved signal peptide of the mutant protein acts as the functional equivalent of the fatty acyl groups normally linked to the N-terminal cysteine residue of the wild-type protein, thereby anchoring the protein in the cell envelope where it exerts its various effects.
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Characterization of the Product of the Cloned fdhF Gene of Escherichia coli
More LessSUMMARY: Random insertions of mini-MudII1734 lac gene fusion bacteriophage were constructed on plasmid pLW06, which carries the fdhF gene coding for benzyl-viologen-linked formate dehydrogenase. They allowed us to limit the size of the gene to a 3 kb fragment and to define its direction of transcription. The identification of the fdhF product as a 85 kDa protein was achieved after expression of derivative plasmids in a maxicell system.
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Genetic Analysis of Conjugational Recombination in Escherichia coli K12 Strains Deficient in RecBCD Enzyme
More LessSUMMARY: Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.
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A Third Multiallelic Mating-type Locus in Physarum polycephalum
More LessSUMMARY: Sexual development (crossing) in Physarum polycephalum occurs when two haploid amoebae fuse to form a diploid plasmodium. The imz locus influences the maximum pH at which crossing can occur. A new allele of imz has been identified, bringing the total number of alleles to three. Contrary to earlier findings, it has been shown that all homoallelic combinations of imz alleles display a similar pH limit for crossing, which is lower than that for imz-heteroallelic combinations. It is concluded that imz is a mating compatibility locus; thus the mating-type system of P. polycephalum comprises three multiallelic loci: matA, matB and imz. It is proposed that imz be renamed matC.
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Analysis of Natural Plasmids of Zymomonas mobilis ATCC 10988
More LessSUMMARY: The six plasmid species of Zymomonas mobilis ATCC 10988 were isolated, separated and purified. Evidence is presented that the 1·6 kb (pZMO1) and 1·9 kb (pZMO2) species can co-migrate on agarose gels, contain single sites for BglII and HindIII, or EcoRI and EcoRV respectively, and are related at the nucleotide level. The 16·7 kb species (pZMO5) appears to be a tetramer and is partially homologous with pZMO1 and pZMO2. The 2·7 kb species (pZMO3) has a single SphI site and does not show sequence homology with pZMO1 or pZMO2. Structural features of the 7·3 kb (pZMO4) and 31·3 kb (pZMO6) molecules are discussed.
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Expression of Dikaryon-specific and Non-specific mRNAs of Schizophyllum commune in Relation to Environmental Conditions and Fruiting
More LessSUMMARY: cDNA clones representing eight mRNAs expressed abundantly in the dikaryon of Schizophyllum commune at the time of fruiting, but very low in the progenitor monokaryons, were used to estimate the concentrations of these mRNAs in cultures of the dikaryon and monokaryon growing under conditions that suppress the formation of fruit-bodies. Such conditions as high atmospheric carbon dioxide and continuous darkness in surface cultures or growth in suspension cultures generally lower the concentrations of these mRNAs in the dikaryon but do not bring them down to the very low levels of these mRNAs in the monokaryons. Therefore the expression of these mRNAs, which depends on the dikaryotic condition, may be required but is not sufficient for the occurrence of fruiting.
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The Isolation of fumB Mutants of Escherichia coli
More LessSUMMARY: Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35·7) is deleted. Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium. These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93·4). A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.
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- Immunology
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Production and Immunological Characterization of a Monoclonal Antibody to Trichophyton quinckeanum: Interaction with Phosphorylcholine-bearing Components
More LessSUMMARY: A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.
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Circulating Antigens and Antibodies in Human and Mouse Dermatophytosis: Use of Monoclonal Antibody Reactive to Phosphorylcholine-like Epitopes
More LessSUMMARY: The presence of circulating antibodies in the sera of patients infected with either Trichophyton concentricum or Trichophyton rubrum, and in the sera of BALB/c mice chronically infected with Trichophyton quinckeanum, was determined by ELISA. High levels of antibody to dermatophyte cytoplasmic antigens were detected both in infected humans and in mice. Partial inhibition of this reaction was observed by pretreatment of the sera with the hapten phosphorylcholine (PC). Moreover, antibodies were shown to have some reactivity with PC when tested by ELISA against PC conjugated to bovine serum albumin. Significant levels of circulating antigen were detected in patients with T. concentricum and T. rubrum infections, but not in uninfected subjects, by an immunoradiometric assay using a monoclonal antibody, Tq-1, which reacts with the PC-like epitopes of dermatophytes. It is possible that this dermatophyte antigen may play a role in modulating the cell-mediated immune responses, which would appear to be defective in most patients with these chronic forms of dermatophytosis.
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- Pathogenicity And Medical Microbiology
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The Potential Protective Effect of Monoclonal Antibodies to Gonococcal Outer Membrane Protein IA
More LessSUMMARY: Hybrid cell lines were derived which produced monoclonal antibodies directed against gonococcal outer membrane protein IA. One antibody, SM101, recognized 24 P.IA-expressing strains out of 25 tested - the rest exhibited relatively less cross-reactivity. Competitive radioimmunoassays revealed that each antibody could effectively inhibit binding of the others, suggesting close proximity of the epitopes recognized. The antibodies were used in assays in vitro to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, the antibodies afforded some protection to epithelial cells challenged with gonococci. They were very effective at bactericidal killing in the presence of complement and, in addition, were opsonic for homologous and heterologous strains. The cross-reacting antibody, SM101, was one of the most effective in both assays. The results show that the conserved epitope on P.IA recognized by antibody SM101 is potentially an effective target on the gonococcal surface for immunoprophylaxis.
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Binding and Cytotoxic Effects of Clostridium botulinum Type A, C1 and E Toxins in Primary Neuron Cultures from Foetal Mouse Brains
More LessSUMMARY: Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 × 102 LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 °C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.
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Preliminary Characterization of the Urease and a 96 kDa Surface-expressed Polypeptide of Ureaplasma urealyticum
More LessSUMMARY: Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.
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Evidence that the Serum Resistance Genetic Locus sac-3 of Neisseria gonorrhoeae Is Involved in Lipopolysaccharide Structure
More LessSUMMARY: The gonococcal chromosome contains a sequence of closely linked genes (for example, sac-1, sac-3, nmp) known or presumed to affect cell envelope structure and which appear to influence susceptibility of gonococci to killing by normal human sera (NHS). Previous work has shown that the serum-resistant isolate FA19, and FA899, a serum-sensitive transformant of FA19, differ in outer membrane protein I (PI) and at the sac-3 genetic locus. However, the sac-3 locus is separable from changes determined by nmp-3, the gene determining PI species. We found that FA 19 and FA899 differ in lipopolysaccharide (LPS) molecular size and in reactivity with a monoclonal antibody which recognizes an LPS (L8) epitope. To address the question of whether the changes in LPS were due to the sac-3 locus, we constructed new transformants of FA 19 using donor DNA prepared from FA899. The new transformants could be divided into three groups: (1) those identical to FA19 in serum resistance (>90% survival at 120 min), in LPS molecular size and in expression of the L8 epitope; (2) those identical to FA899 in serum sensitivity (100% killed at 30 min), in LPS molecular size and in lack of expression of the L8 epitope; (3) those significantly killed by 50% NHS at 120 min, whose LPS molecular size was greater than that of FA 19 but less than that of FA899 and which did not express the L8 epitope. Except for PI there were no differences in other outer-membrane proteins (e.g. PII, PIII, H.8) among these transformants. Studies of PI using monoclonal antibodies which distinguish between PI serovars confirmed that changes in PI were separate from changes in serum resistance or alteration in LPS chemotype. These data indicate that a genetic locus involved in LPS structure appears to be identical with the sac-3 locus, which influences serum susceptibility in Neisseria gonorrhoeae.
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Electrophoretic and Immunochemical Study of the Lipopolysaccharides Produced by Chemostat-grown Escherichia coli 0157
More LessSUMMARY: Two chemically different O-polysaccharides, a low molecular mass form of LPS and core LPS produced by chemostat-grown E. coli O157, were analysed by SDS-PAGE, silver staining and immunoblotting. The reactivities of the different O-polysaccharides with antisera prepared against E. coli O157 grown in batch culture, Salmonella O30 or Brucella abortus were very similar, showing that the O-polysaccharides share at least some antigenic determinants. The reactions of the low molecular mass LPS with the antisera indicated it was semi-rough LPS having one repeat unit of the O-polysaccharide attached to core LPS.
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- Physiology And Growth
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Different Molecular Forms of Invertase in the slime Variant of Neuospora crassa: Comparison with the Wild-type Strain
More LessSUMMARY: Invertase synthesis, regulation and secretion in the wall-less slime variant of Neurospora crassa was studied. Unlike the wild-type, synthesis of the enzyme was not repressed by glucose. This effect was not related to the os mutation harboured by the slime strain, nor to the phenotypic absence of a cell wall. Three molecular forms of extracellular invertase, which varied in size, were detected in the slime strain. These forms were interconvertible, with the equilibrium in favour of the larger form. Polypeptide analysis of the three separated forms revealed that all contained the same glycoprotein with an M r of 97000. This was completely deglycosylated by treatment with endo-β-N-acetylglucosaminidase H (Endo H) to a polypeptide with an M r of 72000. It was concluded that the three interconvertible forms correspond to the monomeric, dimeric and tetrameric states of the enzyme. Three similar forms of invertase, albeit of slightly different electrophoretic mobility, were found in cell-free extracts, cell walls and spent culture medium of the wild-type strain. After Endo H treatment, analysis showed that these forms contained a polypeptide that was equally reactive against anti-Saccharomyces cerevisiae antibodies, and had the same M r, as the polypeptide produced by the slime strain.
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Catabolism of Arginine, Citrulline and Ornithine by Pseudomonas and Related Bacteria
More LessSUMMARY: The distribution of the arginine succinyltransferase pathway was examined in representative strains of Pseudomonas and related bacteria able to use arginine as the sole carbon and nitrogen source for growth. The arginine succinyltransferase pathway was induced in arginine-grown cells. The accumulation of succinylornithine following in vivo inhibition of succinylornithine transaminase activity by aminooxyacetic acid showed that this pathway is responsible for the dissimilation of the carbon skeleton of arginine. Catabolism of citrulline as a carbon source was restricted to relatively few of the organisms tested. In P. putida, P. cepacia and P. indigofera, ornithine was the main product of citrulline degradation. In most strains which possessed the arginine succinyltransferase pathway, the first step of ornithine utilization as a carbon source was the conversion of ornithine into succinylornithine through an ornithine succinyltransferase. However P. cepacia and P. putida used ornithine by a pathway which proceeded via proline as an intermediate and involved an ornithine cyclase activity.
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Coordinated Regulation of Ammonium Assimilation and Carbon Catabolism by Glyoxylate in Saccharomyces cerevisiae
More LessSUMMARY: The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.
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Regulation of Gluconeogenic Enzymes during the Cell Cycle of Saccharomyces cerevisiae Growing in a Chemostat
More LessSUMMARY: Oscillation of the activities of gluconeogenic enzymes (malate dehydrogenase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) was observed during the cell cycle of chemostat cultures of Saccharomyces cerevisiae. Since ethanol is released by the cells at the beginning of the division cycle, its effect on enzyme expression was determined. Pulsing ethanol to a synchronously dividing yeast culture led to a prolongation of the metabolically active phase as indicated by the course of oxygen uptake and carbon dioxide production rates (concomitant ethanol and glucose assimilation). Enzyme activities also remained elevated as long as ethanol was available to the cells. After a substrate shift from glucose to ethanol during cell division, ethanol was used without a lag phase and enzyme induction increased from the level reached at the point of the substrate change. The data confirmed that the small amount of ethanol produced when the cells begin active reproduction acts as an inducer of gluconeogenic enzymes.
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Qualitative Evidence for Expression of Klebsiella pneumoniae nif in Pseudomonas putida
More LessSUMMARY: Pseudomonas putida MT20-3 carrying the Klebsiella pneumoniae nif plasmids pRD1 or pMF250 showed highly O2-sensitive aerobic acetylene reduction on low-N pyruvate or glucose agar. This finding confirms unequivocally that K. pneumoniae nif can be expressed in an obligate aerobe.
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Early Changes in Bacterial Envelopes after Inhibition of Peptidoglycan Synthesis, as Shown by the Use of a Fluorescent Probe
More LessSUMMARY: The probe 8-anilino-1-naphthalene sulphonate (ANS) showed increasing fluorescence with Bacillus subtilis metC lyt-2 cells taken from exponentially growing cultures treated with antibiotics that inhibit cell-wall peptidoglycan synthesis. This increase was due to the probe reaching hydrophobic cell constituents, probably membranes, and started within 30 min of the addition of the antibiotics. This corresponded to the time at which membrane function had been shown to be damaged. The increased fluorescence of the cells with ANS persisted after removal of the antibiotics.
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Negative Chemotaxis of Gametes and Zoospores of Allomyces
More LessSUMMARY: Gametes and zoospores of Allomyces macrogynus and Allomyces arbuscula were repelled by H+, K+, NH4 + or Na+ as well as by Ca2+, Mg2+ and La3+. This negative chemotaxis, which was monitored with a chemotaxis bioassay, occurred no matter what anionic counter-ion was used. The use of a swim-out assay showed that repulsion was similar for all cations and resulted in a band of zoospores that migrated farther into the tube with time. When zoospores allowed to adapt to 10-fold dilutions of 10 mm-KC1 were challenged with 10 mm-KC1 and their motile behaviour monitored in swim-out assays, it was found that the higher the initial concentration of KC1 incubated with the cells, the less repulsion occurred when these cells were challenged with 10 mm-KC1. In addition when zoospores were incubated in a 10 mm solution of one repellent (i.e. KC1, NH4C1 or CaC12) and then challenged in swim-out assays with a 10 mm solution of another repellant (i.e. KC1 or HC1) to determine if the cationic sites of interaction were common or different, the results indicated that the sites were the same since challenging with another cation did not cause zoospore repulsion. The repulsion mechanism was studied further by mixing 10 mm-KC1 and the male pheromone attractant sirenin. These experiments, using the chemotaxis bioassay, showed that in a mixed population of male gametes and zoospores, the male cells were attracted towards the source of the sirenin while all the zoospores were repulsed by KC1. Thus it appears that the mechanism for attraction is different from repulsion or that the attraction mechanism can override the repulsion behaviour.
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An Improved Chemically Defined Medium for Mass Cultures of Tetrahymena: Nutrient Uptake and Growth Regulation
More LessSUMMARY: A chemically defined medium for growing mass cultures of Tetrahymena was developed. The growth characteristics and the effects of variations in the culture conditions and medium constituents were studied. The results show that Tetrahymena can be grown in a completely defined medium with rates and yields nearly comparable to those obtained in non-defined media. In the defined medium, K+ but not Na+, was indispensable for growth; unlike in media containing proteose peptone, the growth rate was independent of the presence of insoluble Fe3+ complexes, but was more dependent on culture temperature. Although Tetrahymena has an absolute requirement for preformed purine and pyrimidine bases in addition to several amino acids, its proliferation and growth were primarily under amino acid control.
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Protoplast Formation and Regeneration of Clostridium acetobutylicum Strain N1-4080
More LessSUMMARY: A method for the production of protoplasts of Clostridium acetobutylicum strain N1-4080 utilizing lysozyme and penicillin G was optimized to yield about 100% protoplasts. Treatment of 109 cells left fewer than 102 that were viable and osmo-resistant. With a mutant strain deficient in autolytic activity, about 1% of the protoplasts regenerated walled cells, whereas most gave rise to stable L-form colonies. Addition of sodium polyanethole sulphonate to the medium improved the regeneration frequency by at least a factor of 10, probably by somehow inhibiting the residual autolytic activity of the strain.
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The Relationship between Glycosyltransferase Production and Membrane Fatty Acid Composition of Streptococcus sanguis NCTC 7865 Grown in the Presence of Protonmotive Force Inhibitors
More LessSUMMARY: The fatty acid composition of Streptococcus sanguis NCTC 7865 was not altered by changing the cation composition (Na+/K+) of the growth medium; glucosyltransferase (GTF; EC 2.4.1.5) also remained constant. In contrast, fructosyltransferase (FTF-S; EC 2.4.1.10) production was reduced by at least 50% in medium with a high Na+ concentration. Growth in the presence of ionophores (gramicidin, nigericin or valinomycin) resulted in an increased proportion of saturated fatty acids, principally octadecanoic acid (C18:0), while the proportion of unsaturated fatty acids, predominantly octadecenoic (C18:1) and hexadecenoic (C16:1) acids, decreased. GTF-S production was reduced in the presence of ionophores whereas FTF-S production was completely abolished. Tween 80 significantly increased both GTF-S production and the proportion of unsaturated fatty acids in the cytoplasmic membrane; FTF-S production was unaltered by Tween 80. The production of GTF-S was inversely proportional to the C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane. It was concluded that FTF-S production is directly influenced by protonmotive force (pmf), whereas GTF-S production is affected more by the physical properties of the cytoplasmic membrane, in particular its fatty acid composition. However, as perturbations in pmf generation can lead to variations in membrane fatty acid composition it can be argued that pmf indirectly influences GTF production by changing the saturated:unsaturated or C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane.
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Correlative Changes of Growth, Pigmentation and Lipid Composition of Dunaliella salina in Response to Halostress
More LessSUMMARY: The growth rate of Dunaliella salina was optimal in the presence of 2·5 to 5% (w/v) NaCl and growth continued up to 30% NaCl. The carotene to chlorophyll ratio gradually increased from about 1·1 at 2·5% NaCl to 7·7 at 30% NaCl and the chloroplast showed some degeneration; however, with 30% NaCl, intact thylakoids were still recognized. The total lipid content of the cells decreased with increasing salinity but the major lipid classes in all extracts were monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols and phosphatidylglycerols. Considerable proportions of diacylglycerotrimethylhomoserines were also present, whereas the proportions of phosphatidylcholines and phosphatidylethanolamines were relatively low. Increasing the salinity of the medium resulted in a large increase in the proportions of constituent linolenic acid (18:3) both in total lipids and in the galactolipids and of palmitoleic acid (16:1) in phosphatidylglycerols. The results are discussed in view of the probable relation of chloroplast lipids to photosynthetic activity, and the fact that the osmoregulator glycerol is produced via photosynthesis.
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Peptidoglycan-bound Polysaccharide Associated with Resistance of Rhizobium loti Strain NZP2037 to Lotus pedunculatus Root Flavolan
More LessSUMMARY: Resistance of Rhizobium loti strain NZP2037 to the prodelphinidin-rich flavolan (condensed tannin) present in the roots of Lotus pedunculatus was associated with the presence of a peptidoglycan-bound flavolan-binding polysaccharide (FBP) in the outer cell membrane of this bacterium. The outer cell membrane of a flavolan-sensitive strain, NZP2213, did not contain this polysaccharide. FBP was extractable in highest concentration from NZP2037 cells during exponential growth of the bacteria. This correlated with exponential-phase cells of NZP2037 showing maximum resistance to flavolan. When added to cultures of NZP2213, FBP afforded a concentration-dependent protection of this strain to the bactericidal effect of the flavolan. ELISA and immunogold labelling of Rhizobium cells using FBP-specific antibodies confirmed the presence of FBP on the outer cell surface of NZP2037 cells and its absence from NZP2213 cells. Co-inoculation of L. pedunculatus with antibiotic-resistant mutants of NZP2037 and NZP2213 resulted in the formation of N2-fixing nodules containing both strains.
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- Systematics
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Comparative 16S rRNA Oligonucleotide Analyses and Murein Types of Round-spore-forming Bacilli and Non-spore-forming Relatives
SUMMARY: The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and ‘B. aminovorans’ with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of ‘B. aminovorans’ the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.
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