- Volume 137, Issue 3, 1991
Volume 137, Issue 3, 1991
- Review Article
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- Biochemistry
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Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis
More LessThe region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the ‘–35’ and ‘–10’ regions of a promoter recognized by RNA polymerase containing σG. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative ‘–10’ heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.
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The subcellular organization of itaconate biosynthesis in Aspergillus terreus
More LessA novel assay method for cis-aconitate decarboxylase (EC 4.1.1.6) has been developed. Differential centrifugation of cell-free extracts of Aspergillus terreus prepared by Novozyme treatment/nitrogen cavitation of itaconate-producing mycelia has shown that cis-aconitate decarboxylase, a key enzyme of itaconate biosynthesis, is localized exclusively in the cytosol. The intracellular localization and maximal activities of nine other enzymes putatively involved in itaconate biosynthesis were examined under both producing and non-producing growth conditions. Two striking differences between growth and production mycelia were observed: (a) cis-aconitate decarboxylase was absent from growing mycelia, and (b) aconitase activity was increased at least two-fold in production mycelia. No significant variations in the maximal activities or intracellular localization were observed for the other enzymes investigated. Mitochondrial and cytosolic isoenzymes of NADP:isocitrate dehydrogenase and malate dehydrogenase were detected by cellulose acetate strip electrophoretic analysis, whereas this analysis detected only single isoenzymes of aconitase and citrate synthase. We therefore propose that in itaconate accumulation cis-aconitate is synthesized in the mitochondrion and then transported to the cytosol where it is converted to itaconate by cis-aconitate decarboxylase.
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Induction of cellulose- and xylan-degrading enzyme systems in Aspergillus terreus by homo- and heterodisaccharides composed of glucose and xylose
More LessSynthetic heterodisaccharides composed of glucose and xylose were tested as inducers of cellulose- and xylan-degrading enzymes in Aspergillus terreus, and the inducing abilities were compared with those of sophorose and xylobiose or their positional isomers. Measurement of secreted and cell-associated enzyme activities revealed that the heterodisaccharides induced the synthesis of the cellulolytic and xylanolytic enzymes, 2-O-?d-glucopyranosyl d-xylose (Glc?-2Xyl) being the most powerful inducer. Sophorose and 2-O-?d-xylopyranosyl d-xylose (Xy1/?-2Xy1), or their positional isomers, selectively induced the synthesis of cellulases and ?xylanases, respectively. An analysis of the extracellular enzymes (which were separated by isoelectric focusing followed by detection using chromogenic and fluorogenic substrates) showed that Glc?-2Xyl initiated the synthesis of specific endo-1,4-?glucanases and specific endo-1,4-?xylanases identical to those produced separately in response to sophorose or Xy1?-2Xy1. Glc?-2Xy1 also induced specific endo-1,4-?glucanases that hydrolysed 4-methylumbelliferyl ?lactoside at the agluconic bond. The results strengthen the concept of separate regulatory control of the synthesis of cellulases and ?xylanases. The results also suggest that mixed disaccharides, composed of glucose and xylose moieties, which may occur in nature, could play an important role in regulating the synthesis of wood-degrading enzymes.
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The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus
More LessMembranes of the acidophilic methylotroph Acetobacter methanolicus contained only b- and c-type cytochrome and a CO-binding b-type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome c and ascorbate/TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent3-12 (1.7 fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (200 mM-NaCl) and stable between pH 4.0 and 6.8. Of the two soluble c-type cytochromes from A. methanolicus only the typical class I cytochrome (cytochrome c H) was a good substrate, as was equine cytochrome c. The oxidase was partially purified by anion-exchange chromatography but further purification proved impossible. The yield with respect to equine cytochrome c oxidation was 18%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome c H and TMPD was lost. Neutral phospholipids had little effect on activity of the oxidase but the charged phospholipids phosphatidylglycerol and phosphatidylserine stimulated activity up to about fourfold. It proved impossible to incorporate the oxidase into active lipoprotein vesicles. During the purification process the pH optimum for oxidation of cytochrome c H was unchanged (pH 5.6) whereas that for oxidation of equine cytochrome changed from pH 9.5 to 7.5 and the sensitivity of the oxidase to azide changed from non-competitive to competitive during the purification process. The partially-purified oxidase contained only b-type cytochrome, some of which was CO-reactive. It is proposed that the oxidase is a cytochrome co type of oxidase that loses its cytochrome c component during the purification process and is only able to oxidize c-type cytochromes if these can be formed into a ‘reconstituted’ cytochrome co with the partially-purified oxidase.
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Superoxide generation during phagocytosis by Acanthamoeba castellanii: similarities to the respiratory burst of immune phagocytes
More LessStarved cultures of the soil amoeba Acanthamoeba castellanii suspended in phosphate-buffered saline exhibit stimulated O2 uptake in response to phagocytosis of either heat-killed yeast or latex beads. This phagocytosis-dependent (cyanide-insensitive) oxidative activity was observed when cells were isolated from either cyanide-stimulated or -inhibited cultures, and was therefore independent of the relative activities of the alternative mitochondrial oxidases known to exist in this organism. The extra O2 consumed during phagocytosis was stoichiometrically converted into O- 2 as detected by the rate of superoxide dismutase-inhibitable cytochrome c reduction. Phagolysosomal membranes isolated after uptake of latex beads were enriched in a b-type cytochrome which was spectrally similar to that present in immune phagocytes. Thus, the biochemical events during phagocytosis by either A. castellanii or immune phagocytes appear similar, suggesting that the ‘respiratory burst’ enzyme(s) responsible for O- 2 generation in these two cell types is structurally related.
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Occurrence of a novel yeast enzyme, l-Lysine e-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans
More LessThe yeast Candida albicans is able to utilize l-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of a-aminoadipate-d-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the e-group of l-lysine was found in this yeast. The enzyme, l-lysine e-dehydrogenase, is strongly induced in cells grown on l-lysine as the sole nitrogen source. The enzyme is specific for both l-lysine and NADP+. The K m values were determined to be 0.87 mM for l-lysine and 0071 mM for NADP+. An apparent M r of 87000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 °C under our assay conditions.
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Protein methylases in Trypanosoma brucei brucei: activities and response to dl-a-difluoromethylornithine
More LessProtein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-l-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent K m for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent K m for S-AdoMet of 8.4 μM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-l-homocysteine and its analogue sinefungin with apparent K i values of 12.9 and 1.6 μm, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.
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- Biotechnology
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Improvement of bacterial β-glucanase thermostability by glycosylation
More LessThe relationship between enzyme stability and glycosylation was examined for two different Bacillus (1,3-1,4)-β-glucanases following expression of the corresponding genes in Escherichia coli and in Saccharomyces cerevisiae. Both of the (1,3-1,4)-β-glucanases secreted from yeast cells were glycosylated and a pronounced difference in the type and extent of glycosylation was observed. Thermostability analysis of the glycosylated enzymes and their unglycosylated counterparts synthesized by E. coli disclosed a substantially higher thermotolerance of the glycosylated enzymes. At 70 °C the half-life of the glycosylated form of B. macerans (1,3-1,4)-β-glucanase was 26 min, as compared to 10 min for the unglycosylated form of the enzyme. Using the same conditions, the half-life of the B. amyloliquefaciens-B. macerans hybrid (1,3-1,4)-β-glucanase was 5 min for the unglycosylated enzyme and about 100 min when the enzyme was glycosylated.
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Factors affecting the normal and branched-chain acyl moieties of teicoplanin components produced by Actinoplanes teichomyceticus
More LessTeicoplanin, a glycopeptide antibiotic produced by Actinoplanes teichomyceticus, comprises five main components, denoted T-A2 to T-A2-5, differing in the structure of their acyl side chain, which is linear in T-A2-1 and T-A2-3 and branched in the other components. Production of T-A2-1, characterized by a linear C10:1 acyl moiety, is entirely dependent on the presence of linoleate in the fermentation medium. Addition to the medium of oleic acid esters at 2 g l-1 increases the yields of T-A2-3, characterized by a linear C10:0 acyl chain, about threefold. The antibiotic linear side chains thus appear to originate from C18 unsaturated acid by β-oxidation degradation. The percentage of T-A2-2, T-A2-4 and T-A2-5, bearing the iso-C10:0, anteiso-C11:0 and iso-C11:0 acyl moieties, respectively, is strongly influenced by the presence in the medium of the amino acids known to be precursors of branched-chain fatty acids. Thus, valine increases the production of T-A2-2 whereas isoleucine or leucine increase the relative yields of T-A2-4 or T-A2-5, respectively. Analysis of the total cell lipids upon addition of the same amino acid shows corresponding increases in the proportion of the iso-C16:0, iso-C15:0 or anteiso-C17:0. A mutant A. teichomyceticus strain, which produces a novel teicoplanin with a linear C9:0 chain, differs from the wild strain in the presence of the linear C17:1 acid in its lipids. The relative incorporation of [14C]acetate into teicoplanin acyl moieties is substantially lower when this precursor is added to grown A. teichomyceticus mycelium rather than at the time of inoculation. The results suggest that teicoplanin branched acyl moieties also originate from β-oxidation degradation of cellular long-chain fatty acids.
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- Development And Structure
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Cell wall and autolytic system of Lactobacillus helveticus ATCC 12046
More LessThe cell wall of Lactobacillus helveticus was found to have a three-layered structure when thin sections of whole cells or isolated cell walls were stained by various procedures and examined by electron microscopy. The main feature of the composition of isolated cell walls was a high protein content (about 48% of the cell wall dry weight) with a predominant 52 kDa protein. Peptidoglycan and what was considered as teichoic acids accounted for 36% and 14%, respectively, of the cell wall dry weight. The 52 kDa protein was apparently unglycosylated, located in the outer layer and noncovalently bound to the inner layers. Peptidoglycan was mostly assigned to the innermost layer. The conditions leading to autolysis of whole cells or isolated cell walls were determined, as well as the specificity of the autolysin involved in cell wall degradation.
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- Ecology
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Attachment and replication of Pseudomonas aeruginosa bacteriophages under conditions simulating aquatic environments
More LessBacterial viruses are important in regulating bacterial population densities in natural aquatic environments, but the dynamics of bacteriophage-bacterial interactions in nature are poorly understood. In this study, the attachment and replication of three Pseudomonas aeruginosa bacteriophages (one temperate and two virulent) were investigated under conditions similar to those found in nature. Attachment and replication of bacteriophages were not impaired at host-cell densities equal to or lower (< 105 c.f.u. ml-1) than those frequently found in aquatic environments when the host cells were physiologically competent to allow phage growth. Attachment (45–93%) to either actively growing or starved cells was not impaired in river water, indicating that attachment is efficient in natural freshwater habitats. However, the replication of bacteriophages was significantly altered in starved cells in river water: the latency period was extended (broth, 70–110 min; river water, 110–240 min), and the burst size was reduced (broth, 27–65; river water 5–7). The findings of this study indicate that phages are likely to affect microbial ecology significantly in freshwater ecosystems.
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- Genetics And Molecular Biology
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Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207
More LessChlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1·5 kb labelled DNA probe containing the 3′ region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8·5 kb BglII fragment containing the complete MOMP gene was cloned into λEMBL3. Two hybridizing EcoRI fragments were sub-cloned into the λZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.
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Separate cloning and expression analysis of two protein components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. C8S3
More LessThe two protein components, II and III, of the 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 were cloned separately into Escherichia coli. Component II was obtained on plasmid pCBSII, containing a 3.0 kbp HindIII fragment, and component III on plasmid pCBSIIIb, containing a 1.3 kbp SalI/PstI fragment. The identities of the two components were confirmed by comparison with the authentic components from Pseudomonas sp. CBS3. Both components were expressed constitutively in E. coli. Neither component alone showed dehalogenating activity. Only in the mixture of crude extracts from both clones was 4-chlorobenzoate dehalogenase detectable. The specific activities in E. coli crude extracts were 2.9 mU (mg protein)−1 for component II and 3.5 mU (mg protein)−1 for component III. Expression analysis by minicell experiments revealed a single polypeptide chain of 29 kDa for component II and of 31 kDa for component III.
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Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase
More LessTwo mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2·6-fold and 4·7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.
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A chromosomal hotspot for multiple rearrangements associated with genetic instability of Streptomyces ambofaciens DSM 40697
More LessGenetic instability in Streptomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.
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Isolation and characterization of cDNA clones encoding polypeptides related to a Dictyostelium discoideum cyclic AMP binding protein
More LessBy screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.
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Analysis of the Serratia marcescens proBA operon and feedback control of proline biosynthesis
More LessThe proBA operon, coding for γ-glutamyl kinase (GK) and γ-glutamyl phosphate reductase (GPR), was cloned from the chromosome of the wild-type strain of Serratia marcescens Sr41. From our sequence data the proB (1101 bp) and proA (1472 bp) genes were shown to code for two proteins of M r 39169 and 44640, respectively. Analysis of expression of the lacZ structural gene fused with the proBA promoter showed that the proBA operon is not subject to proline-mediated feedback repression. Amplification of the proBA operon enabled us to determine GK activity, which was inhibited in the presence of a low concentration of l-proline. Comparison of the amino acid sequences of the S. marcescens GK and GPR proteins with those of the GK and GPR proteins from E. coli revealed extensive similarities.
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Cloning of a DNA fragment encoding the 5′-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340
Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using λgt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.
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Reference collection of strains of the Salmonella typhimurium complex from natural populations
A collection of 72 reference strains of the Salmonella typhimurium complex of clones recovered from a variety of hosts and environmental sources in diverse geographic locations has been established for use in studies of variation in natural populations. Included are strains of the serovars S. typhimurium, S. saintpaul, S. heidelberg, S.paratyphi B (including variety java) and S. muenchen. The strains, which have been characterized by enzyme electrophoresis for allelic variation in 24 chromosomal structural genes and represent 48 distinctive multilocus genotypes (electrophoretic types or ETs), exemplify the full range of genotypic variation in the S. typhimurium complex. Evolutionary genetic relationships among the ETs are indicated in a phylogenetic tree generated by the neighbour-joining method from a matrix of Nei's standard genetic distance.
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Identification of four unique clones encoding 10 kDa proteins from Bacillus that cause phenotypic complementation of a phoA mutant strain of Escherichia coli
More LessA number of clones have been isolated from two Bacillus species which complement the PhoA- phenotype of Escherichia coli mutants under conditions that induce the expression of alkaline phosphatase (APase). These clones were initially thought to carry XPases because the transformed host could hydrolyse a common APase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate). The sequences of the open reading frames responsible for the phenotypic complementation showed no sequence similarity to APases of E. coli, human (bone-liver-kidney, intestinal or placental) or Bacillus. Therefore, these clones were designated as XPA (for X Phosphatase Activity) clones. Four of the clones encoded small (10 kDa), basic, hydrophobic proteins. Two of these, xpaB from B. subtilis 168 and xpaL2 from B. licheniformis MC14, shared 62% identity at both the DNA and the predicted amino acid sequence level. The fact that homologues from two Bacillus strains were cloned indicated that the screen was specific, but not for APase genes. It is clear that phenotypic complementation with cloned DNA from another genus does not ensure the identification of an APase gene. Possible mechanisms for the abnormal phenotypic complementation are discussed.
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Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA
More LessIn Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism. In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level. To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA. The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2. However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression. These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene.
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Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue-resistant mutants
More LessControl of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and a-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.
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- Immunology
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A cell surface/plasma membrane antigen of Candida albicans
More LessAntibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with β-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzymes (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope.
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- Pathogenicity And Medical Microbiology
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The cytotoxin of Actinobacillus pleuropneumoniae (pleurotoxin) is distinct from the haemolysin and is associated with a 120 kDa polypeptide
More LessMutants of Actinobacillus pleuropneumoniae strain HK 361 (serotype 2) were isolated which were deficient in type II (Ca2+-dependent) haemolysin activity (Hly-). Some of the Hly- mutants secreted a potent, heat-labile extracellular cytotoxic activity against porcine alveolar macrophages. Comparison of cell-free culture supernatant from the parent strain and some Hly- mutants by SDS-PAGE and immunoblotting revealed the loss of a major extracellular polypeptide of 109 kDa. Two Hly- mutants which in addition failed to secrete a 120 kDa polypeptide produced no extracellular cytotoxic activity, suggesting that the 120 kDa protein was the cytotoxin. Antiserum raised to the culture supernatant from a Hly- mutant lacking the 109 kDa polypeptide recognized the 120 kDa band, but not the 109 kDa band, in immunoblots and neutralized the cytotoxic activity, but not the haemolytic activity, of A. pleuropneumoniae. The 120 kDa polypeptide and extracellular cytotoxic activity were widespread among A. pleuropneumoniae strains, but absent from related bacterial pathogens of the pig: Actinobacillus suis, Haemophilus parasuis and Pasteurella multocida. A clear correlation was found between the presence of the 120 kDa polypeptide and cytotoxic activity in culture supernatants. The cytotoxic activity of all the strains tested was neutralized by antibody to the Hly- extracellular material and by convalescent pig serum. It is proposed that the 120 kDa polypeptide represents the cytotoxin of A. pleuropneumoniae, that it is distinct from the haemolysin, and that it be termed pleurotoxin.
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Molecular characterization of a new plasmid-encoded SHV-type β-lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae
More LessBetween 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMPI. The MIC values for cefotaxime of the original isolates and the transconjugants were > 128 mg I-1 and 64 mg I-1. respectively, Isoelectric focusing of protein preparations from the transconjugants showed a β-lactamase with a pl of 7.6. A 3.6 kb BamHl fragment containing the β-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and Pst l subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3′ noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5′ upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the β-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available β-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant β-lactamase of the SHV-type and name it SHV-2a.
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- Physiology And Growth
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Permeability of dormant spores of Bacillus subtilis to malachite green and crystal violet
More LessThe permeability of dormant spores of Bacillus subtilis to malachite green (MG) and crystal violet (CV) was examined by using potassium trichloro(η2-ethylene)platinum(II) (KTPt) as an electron-opaque marker for the dyes. The spores were treated with the dyes and other substances at 30 °C for 30 min or at 80 °C for 5 min. When the spores were incubated in 50 mM-MG solution at 30 °C and in 50 mM-CV solution at 30 °C or 80 °C, many small electron-dense precipitates, which were chemical complexes of dyes and platinum, were seen, mainly around the boundary between the inner and outer coat regions. The spores treated under the above conditions were not stained. Treatment with 50 mM-MG alone or a mixture of 25 mM-oxalic acid and 50 mM-CV at 80 °C made the spores stainable and dye-TPt precipitates were observed mainly in the outer pericortex region. Pretreatment with 25 mM-oxalic acid and 5% (v/v) phenol at 80 °C followed by 50 mM-CV treatment at 30 °C gave the same results as above. It was considered from these results that the inner coat itself might function as the primary permeability barrier to MG and CV, and that a secondary barrier to the dyes might exist around the cortex region.
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Chitin synthesis in Fusarium graminearum and its inhibition by edifenphos (Hinosan)
More LessMembrane-bound chitin synthase from Fusarium graminearum A3/5 had a pH optimum of 6·5, an apparent temperature optimum of 30 °C and an apparent K m for UDP-N-acetylglucosamine (UDP-GlcNAc) of 2·5 mm. Digitonin-solubilized chitin synthase had a lower (about 25% of the original activity) and more variable activity than the membrane-bound chitin synthase from which it was prepared. The activity of solubilized chitin synthase was stabilized by incorporating Allosamidin (a chitinase inhibitor) into the reaction mixture, and an approximately 3·6-fold increase in activity was observed when the reaction mixture contained dimyristoylphosphatidylcholine. Concentrations of the organophosphorus fungicide edifenphos (Hinosan) up to 25 μm had no effect on the in vitro activity of membrane-bound chitin synthase, but between 25 and 145 μm, chitin synthase activity decreased with increase in fungicide concentration. Edifenphos caused non-competitive inhibition of chitin synthase activity with an apparent K i of 50 μm. Membrane-bound chitin synthase preparations from cultures of F. graminearum grown in 250 μm-edifenphos had a much lower in vitro activity than preparations from cultures grown in the absence of the fungicide. Pre-growth of F. graminearum in 250 μm (but not 80 μm) edifenphos also caused a reduction in incorporation of [3H]GlcNAc into chitin in vivo (washed biomass was incubated in [3H]GlcNAc-containing medium lacking fungicide).
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Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae
More LessKlebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrte specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.
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Glycerol catabolism in Aspergillus nidulans
More LessGlycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific introduction. Biochemical and genetical analyses show that dihydroxyacetone and d-glyceraldehyde are converted into glycerol and then catabolized by the same pathway, d-Glyceraldehyde can be reduced by NADP+-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.
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Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae
More LessGlutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of γ-glutamyltranspeptidase (γ-GT), the enzyme initiating GSH degradation. However, γ-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the γ-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of γ-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and γ-cystathionase seemed different from those playing a role in γ-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.
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Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid
More LessPlasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4–50 mg I–1 (0·03–0·35 mM), pH 4·0] was 1·5-fold higher than that in cells grown in its absence. The K m for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6·5 with octanoic acid at 100 mg I–1 or less (2·4 mg acid form plus 97·6 mg octanoate ion I–1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.
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- Plant-Microbe Interactions
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Mechanism of action of the phytotoxin syringomycin: a resistant mutant of Saccharomyces cerevisiae reveals an involvement of Ca2+ transport
More LessThe bacterial phytotoxin syringomycin affects plasma-membrane-associated functions of plants and yeast. These include increases in transmembrane K+, H+ and Ca2+ fluxes and membrane potential. Mutants of Saccharomyces cerevisiae resistant to growth inhibition by syringomycin were isolated and characterized. Many of the mutant isolates were unable to grow in yeast extract/peptone/dextrose medium supplemented with 400 mM-CaC12 which permitted the growth of the parent strain (8A-1B). Genetic analyses of one of these mutants, strain R4-3G, showed a single recessive mutation that simultaneously led to Ca2+-sensitivity and syringomycin-resistance. R4-3G had higher net 45Ca2+ uptake rates than strain 8A-1B and higher intracellular Ca2+ levels in medium containing 1 mM-CaC12. The altered 45Ca2+ uptake rates of the mutant were not influenced by syringomycin and were not related to altered capabilities for Ca2+ efflux. R4-3G had similar syringomycin-stimulated increases in K+ efflux but lower syringomycin-stimulated increases in membrane potential than 8A-1B. It is concluded that Ca2+ transport is important in the response of yeast to syringomycin and that the toxin-stimulated membrane potential increase, but not K+ efflux, is closely associated with Ca2+ transport.
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- Systematics
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Differentiation among strains and serotypes of Bacillus thuringiensis by M13 DNA fingerprinting
More LessThe inter- and intraserotypic variations of Bacillus thuringiensis were studied by M13 DNA fingerprinting. Strain-specific patterns were obtained. The degree of homology was evaluated on the basis of pairwise comparisons and calculation of similarity indexes. Some strains belonging to the same serotype showed highly similar patterns, but others differed significantly. A high degree of polymorphism was established among the serotypes. These results provide evidence that the classification of B. thuringiensis strains on the basis of flagellar antigens does not always adequately reflect their genetic relatedness. DNA fingerprinting could help in future numerical taxonomic analysis of this species.
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