- Volume 139, Issue 3, 1993
Volume 139, Issue 3, 1993
- Biochemistry
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Purification and Properties of an Extracellular Aminopeptidase from Streptomyces Lividans 1326
More LessStreptomyces lividans was investigated for extracellular proteinases. The major extracellular proteinase activity detected under all conditions tested was l-leucine aminopeptidase activity. Slight extracellular l-proline aminopeptidase activity was also detected. No clear evidence for the presence of serine proteinases in S. lividans culture broths was found using several different methods. The major extracellular proteinase of S. lividans, i.e. the l-leucine aminopeptidase, was purified 33-fold to homogeneity. The purified enzyme was found by SDS-PAGE to have an M r of 34000. The purified enzyme had a final specific activity of 3·6 units mg−1, a K m for l-leucine-p-nitroanilide of 300 μm and a V max of 4·2 μmol min−1 mg−1. The pure enzyme did not exhibit proteolytic activity on azocasein or l-proline-p-nitroanilide, substrates for other proteinase activities observed in crude extracts of S. lividans.
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Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum
More LessA genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for xanthan polysaccharide synthesis (xps) was undertaken. Seven xps DNA regions were isolated after conjugation of chemically induced non-mucoid mutants with a genomic library of X. campestris DNA. No overlapping segments between regions were detected, based on physical mapping, indicating the unlinked character of these regions. Clones complementing several different mutants belonging to the same region contained overlapping segments of X. campestris chromosomal DNA. Complementation and biochemical analysis, and DNA mapping were used to identify and characterize xpsIII, IV and VI DNA regions. Mutants in these three regions were able to synthesize both lipid intermediates and xanthan gum in vitro when sugar nucleotides were provided as substrates. HPLC analysis of the intracellular sugar nucleotide content showed that the XpsIII group comprises two different classes of mutants: XpsIIIA, defective in UDP-glucose, UDP-glucuronic acid and GDP-mannose, and XpsIIIB, defective in GDP-mannose. XpsIV mutants were defective in UDP-glucose and UDP-glucuronic acid, and XpsVI mutants were defective only in UDP-glucuronic acid. Analysis of enzyme activities involved in the synthesis of UDP-glucose, GDP-mannose and UDP-glucuronic acid indicated that the xpsIIIA region affects the activity of the phosphoglucomutase/phosphomannomutase enzyme, and the xpsIIIB region affects the mannoisomerase/ phosphomannoisomerase activities. The xpsIV mutations affect the activity of the UDPG-pyrophosphorylase enzyme, and the xpsVI mutations affect the activity of the UDPG-dehydrogenase enzyme.
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Positional distribution of fatty acids, and molecular species of polar lipids, in the diatom Phaeodactylum tricornutum
More LessEach of the four main polar lipids from Phaeodactylum tricornutum UTEX 640, monogalactyosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylcholine (PC), was separated into its molecular species by reverse-phase HPLC, and the positional distribution of fatty acids in each species was determined. Numbers of separated peaks in each lipid class were: MGDG, 10; DGDG, 7; SQDG, 10; and PC, 11. Eicosapentaenoic acid (EPA) was present in 20 of the 45 molecular species but predominated in the MGDG and DGDG classes, where 13 of the proposed structures contained EPA. EPA was always located in the sn-1 position except in two lipid species, MG5 and PC2, where it was present at both the sn-1 and sn-2 locations. The predominant polar lipid molecular species found in P. tricornutum UTEX 640 were (mg total fatty acids per g dry weight of biomass) 20:5–16:4–MG, 41·2; 20:5–16:1–DG, 21·0; 16:1–16:1–SQ, 18·2; and 20:5–16:1–MG, 18·0. [Structure indicates fatty acid at sn-1 position - fatty acid at sn-2 position-carbohydrate component at sn-3 position of the glycerol molecule. Abbreviations: 20:5, EPA; 16:4, hexadecatetraenoic acid; 16:1, palmitoleic acid; MG, monogalactose; DG, digalactose; SQ, sulphoquinovose.]
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Further Characterization of the Assimilatory Nitrate Reductase from the Yeast Candida Nitratophila
More LessNitrate reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b 557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.
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Purification and Partial Characterization of Acid Phosphatase from Candida Lipolytica
More LessNon-specific acid phosphatase from Candida lipolytica cells was purified 111-fold by chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 and Sepharose 4B. The enzyme is a glycoprotein containing 67% neutral sugars. The molecular mass of the highly purified acid phosphatase was found to be approximately 95 kDa by both SDS-PAGE and gel filtration. The pH and temperature optima were 5·8 and 55 °C, respectively. The enzyme was stable at pH values between 3·5 and 5·5 and at temperatures up to 60 °C. The purified phosphatase had a K m value of 364 mm for p-nitrophenyl phosphate and showed broad substrate specificity.
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Investigation of a Killer Strain of Zygosaccharomyces Bailii
More LessThe yeast Zygosaccharomyces bailii strain 412 was found to liberate a killer toxin (KT412) lethal to sensitive strains of Saccharomyces cerevisiae and Candida glabrata. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular protein was purified by gel filtration and ion-exchange chromatography. Gel filtration and SDS-PAGE of the electrophoretically homogeneous killer protein indicated an apparent molecular mass of 10 kDa. The killer toxin KT412 is probably not glycosylated since it did not show any detectable carbohydrate structures. KT412 was bound to sensitive but not to resistant yeast cells. The mannan, and not the glucan, fraction of the cell wall of the sensitive yeast was the primary target for the killer toxin binding. The killer strain Z. bailii 412 contained three double-stranded RNA plasmids of 1·9, 2·9 and 4·0 kb. Curing by cycloheximide resulted in the concomitant loss of killer activity and the 1·9 kb dsRNA species that is therefore regarded as equivalent to the killer-toxin-coding M-plasmids of S. cerevisiae.
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Characterization and Complementation of Mutants of Methylophilus Methylotrophus that have Thermolabile Forms of Proteins Involved in C1 Metabolism
More LessTwo temperature-sensitive mutants of Methylophilus methylotrophus have been isolated and characterized. The first, NH-4, had a temperature-sensitive defect in methylamine oxidation and was unable to utilize methylamine as sole carbon and nitrogen source at 42 °C. The activities of the methylamine oxidation system and methylamine dehydrogenase in cells grown on methylamine at 30 °C were much more thermolabile than those of the wild-type. Furthermore, the affinity of the mutant enzyme for methylamine was lower than that of the wild-type enzyme. These results suggest that NH-4 produces a mutant enzyme with an altered conformation which is more susceptible to thermal denaturation than the wild-type enzyme. Surprisingly, this mutant could grow at 42 °C on media containing methylamine if an alternative carbon or nitrogen source was available. The methylamine oxidation system of whole cells grown under these conditions was not inactivated at 42 °C. The second mutant, NH-7, was unable to grow at the restrictive temperature on media containing either methanol or methylamine as sole carbon source. It contained thermolabile forms of methanol and methylamine dehydrogenases and cytochrom c L. This phenotype could be due to a mutation in a gene essential for the production of mature forms of these periplasmic proteins, which are involved in C1 metabolism. Cosmid pAD833, which has previously been shown to carry genes involved in methanol oxidation (mox genes), complemented both NH-4 and NH-7. Subcloning indicated that the gene which complemented NH-7 was within a 3 kbp region which contained at least two mox genes. This region was near an 8 kbp region containing the gene which complemented mutant NH-4.
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A Triacyltrehalose Containing 2-Methyl-Branched Unsaturated Fatty Acyl Groups Isolated from Mycobacterium Fortuitum
More LessA variety of glycolipids were found in a collection of strains of Mycobacterium fortuitum and two patterns were established by thin-layer chromatography. A compound present in all the strains studied was identified as 2,3,4-triacyltrehalose and its overall structure determined by infrared spectroscopy, nuclear magnetic resonance spectroscopy and gas-liquid chromatography-mass spectrometry. Fatty acyl groups present in this molecule were identified as tetradecanoyl, hexadecenoyl, hexadecanoyl, octadecenoyl, octadecanoyl and a variety of 2-methyl-branched unsaturated (α-methyl, α,β-unsaturated) acyl groups. The 2-methyl-branched compounds ranged from 17 to 21 carbon atoms, the most abundant being 2-methyloctadecen-2-oyl. Trehalose was the only sugar detected in the glycolipid. This substance immunoreacted with IgG and IgM present in sera from tuberculosis patients, as demonstrated by an enzyme-linked immunosorbent assay.
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- Biotechnology
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Changes in Lipid Composition and Arachidonic Acid Turnover during the Life Cycle of the Yeast Dipodascopsis Uninucleata
More LessUngerminated ascospores of Dipodascopsis uninucleata contained 18 times more lipid (5·5% dry wt) than germinated cells; the lipid comprised 58% (w/w) glycolipids, 28% (w/w) neutral lipids (mainly triacylglycerols) and 14% (w/w) phospholipids (mainly phosphatidylcholine and phosphatidylethanolamine). During germination the absolute amounts of all three lipid fractions fell sharply but, during the subsequent initiation of hyphal growth, the amount of phospholipids increased. As these hyphae began to differentiate for the sexual stage of the life cycle, the amount of neutral lipid then increased. The fatty acyl groups of the glyco-, neutral and phospholipid fractions throughout the life cycle were mainly palmitate (16:0), oleate (18:1) and linoleate (18:2). The percentage of 16:0 remained constant during the life cycle while the relative amounts of 18:2 plus α-linolenate (18:3) in the glyco-,neutral and phospholipid fractions first increased during initiation of growth and then decreased during the onset of differentiation. The opposite trend occurred with 18:1. When [3H]arachidonic acid (ARA) and [1-14C]18:1 were fed separately to D. uninucleata, both were rapidly incorporated into phospholipids. Highest incorporation of ARA was in the growth phase; during the onset and remainder of the differentiation phase, the amount of ARA decreased in this fraction. Incorporation of 18:1 increased during growth and differentiation, with a significant proportion (49% to 57%) being incorporated into triacylglycerols compared to a much smaller proportion (12% to 17%) of ARA.
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- Genetics And Molecular Biology
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Primary Structure, Partial Purification and Regulation of Key Enzymes of the Acetyl Cycle of Arginine Biosynthesis in Bacillus Stearothermophilus: Dual Function of Ornithine Acetyltransferase.
A 3·4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M r of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli argB mutants transformed with the 3·4 kb fragment on a plasmid. A polypeptide band of M r 27000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M r 43349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M r 110000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.
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Molecular and Enzymological Evidence for Two Classes of Fumarase inBacillus Stearothermophilus(var.Non-Diastaticus)
More LessThe gene (fumA Bst) encoding an oxygen-labile fumarase of Bacillus stearothermophilus has been cloned and sequenced. The structural gene (1542 bp) encodes a product (FumABst) of M r 56788 containing 514 amino acid residues. The amino acid sequence is 23% identical (37% similar) to FumA and FumB, the labile [4Fe-4S]-containing fumarases (Class I enzymes) of Escherichia coli. It exhibits no significant similarity to FumC and CitG, the stable fumarases (Class II enzymes) of E. coli and Bacillus subtilis (respectively). Enzymological studies indicated that FumABst resembles the iron-sulphur-containing fumarases in being dimeric (M r 2 × 58500), oxygen labile and partially reactivated by Fe2+plus DTT. The fumA Bst gene is the first gene encoding a Class I fumarase to be characterized in any organism other than E. coli. Enzymological and DNA-hybridization studies further indicated that B. stearothermophilus resembles E. coli in containing an oxygen-stable fumarase (Class II enzyme). Sequence comparisons revealed significant similarities between the Class I fumarases and the products of adjacent open-reading frames (orfZ1 and orfZ2) located upstream of the macromolecular synthesis operon (rpsU-dnaG-rpoD) at 67 min in the E.coli linkage map. Located downstream of fumA Bst, there is an unidentified gene (orf2), which is homologous to the rhizobial nodB genes involved in the initiation of root nodule formation.
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Putrescine Oxidase of Micrococcus Rubens: Primary Structure and Escherichia Coli
More LessThe flavin adenine dinucleotide (FAD)-containing putrescine oxidase of Micrococcus rubens catalyses the oxidative deamination of putrescine. The amino acid sequences of the NH2-termini of the mature enzyme and lysyl-endopeptidase-generated fragments were determined for preparation of synthetic oligonucleotides as hybridization probes for cloning. A 4·4 kb BamHI fragment which contained DNA sequences hybridizing to the probes was cloned in pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 480 amino acids (M r 52000) and contains an FAD-binding consensus sequence at its NH2-terminal portion. In front of the transcriptional start point, which is 28 bases upstream of the initiation codon as determined by primer extension, −35 and −10 sequences similar to typical prokaryotic promoter consensus sequences are present. E. coli JM109 containing the putrescine oxidase gene just downstream of the lac promoter in pUC18 produced a large amount of this protein when grown at 37 °C but in the enzymically inactive form of inclusion bodies. However, cultivation of the recombinant E. coli cells at temperatures below 30°C led to production of active enzyme (20 times as much as produced by the original M. rubens strain).
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Properties of Genes Involved in the Control of Isocitrate Lyase Production in Aspergillus nidulans
More LessThe interaction between genes of Aspergillus nidulans conferring constitutive synthesis of isocitrate lyase (iclcA and iclcB) and fluoroacetate resistance (facB) has been investigated. Although facB mutants are unable to induce the glyoxylate cycle enzyme isocitrate lyase in response to acetate as sole carbon source, this phenotype was suppressed in recombinants of the type iclc;facB. The iclcA and iclcB mutations do not alter significantly the activities of eight enzymes of intermediary metabolism assayed. We conclude that the iclc genes are probably bona fide isocitrate lyase regulatory genes.
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Investigation of Thermolabile Variants of the Methanol and Methylamine Dehydrogenases of Methylophilus Methylotrophus and the Effect of an 11·5 kbp Region of the Chromosome on the Stability of these enzymes
More LessThe thermal stabilities in vitro of the methylamine and methanol dehydrogenases of Methylophilus methylotrophus varied, depending on the conditions in which the organism was grown. Methylamine dehydrogenase activity was more stable in extracts of cells grown on methylamine plus NH+ 4 or on methanol plus methylamine than when methylamine provided the sole carbon and nitrogen source. In contrast, the methanol dehydrogenase activity in extracts of methylamine-grown cells was more stable than that from cells grown on medium containing methanol with either NH+ 4 or methylamine as the nitrogen source. The stability of thermolabile forms of the dehydrogenases present in extracts of mutants which have temperature-sensitive defects in the oxidation of C1 compounds also varied with the growth conditions. When cosmid pAD833, which complemented these temperature-sensitive mutants, was present during growth of the wild-type strain on methylamine, the thermal stabilities of both methylamine and methanol dehydrogenases increased. However, the effects of subclones of pAD833 were complex and our results indicate that the DNA region carried on this cosmid encodes gene products which can both increase and decrease the in vitro thermal stability of enzymes involved in C1 metabolism.
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A Comparison of the Multiple Alleles of xylS Carried by TOL Plasmids pWW53 and pDK1 and its Implications for their Evolutionary Relationship
Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylS pWW0. xylS1 pWW53 is 99% identical to xylS pWW0 and is located relative to the single copy of xylR pWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3 pWW53 is 87% identical to the xylS pWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2 pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1 pWW53 and xylS3 pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1 pDK1 is functional and is a hybrid gene: its 5� end and the upstream sequences are highly homologous to both xylS pWW53 and xylS pWW0, whereas its 3� end is identical to xylS3 pWW53. The sequence of xylS2 pDK1 is identical to that of the anomalous truncated xylS2 pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.
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A Mobilizable Shuttle Vector for the Cyanobacterium Plectonema Boryanum
More LessPlasmid pSUP5011 contains a pMB1 origin of replication, an origin of transfer (oriT), and genes encoding resistance to the antibiotics ampicillin, chloramphenicol and kanamycin. pSUP5011 was conjugally mobilized from Escherichia coli into the non-heterocystous, filamentous, nitrogen-fixing cyanobacterium Plectonema boryanum UTEX 594 in the presence of a helper plasmid, RP4. Transconjugant cyanobacteria selected for resistance to kanamycin, ampicillin and chloramphenicol showed a variety of DNA rearrangements in pSUP5011. One such plasmid continued to show characteristic rearrangements following subsequent transfers into the cyanobacterium. A stable plasmid useful as a shuttle vector was isolated.
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Cloning and Sequencing of the Gene which Encodes the Highly Inducible Acetamidase of Mycobacterium Smegmatis
More LessThe acetamidase of Mycobacterium smegmatis NCTC 8159 was purified, and the sequences of its amino-terminus and of two peptides obtained by proteolysis of the protein were obtained. A DNA fragment including the amidase structural gene was cloned in Escherichia coli, using oligonucleotide probes designed on the basis of the peptide sequences and a codon usage table calculated from published sequences of nine protein-antigen-encoding genes of the Mycobacterium tuberculosis complex. Sequence analysis of the cloned DNA revealed that the amidase gene encoded 406 amino acid residues. The nucleotide sequence close to and upstream of the amidase gene contained a probable ribosome-binding site but no identifiable promoter sequences. Three additional potential open-reading frames were found upstream of and very close to the amidase gene, with consensus ‘−35’ and ‘−10’ promoter sites between the first and second of these. It is hoped that the highly inducible expression of the acetamidase gene can be exploited to allow regulated expression of other genes cloned in mycobacteria.
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Characterization of Two Different Types of Resistance Genes Among Producers of Fortimicin-Group Antibiotics
More LessFortimicin-A (FTM-A; astromicin)-resistance genes (fmr genes) isolated from six producers of the FTM-group of antibiotics were analysed. These genes could be classified into two types by the resistance profiles to aminoglycoside antibiotics and by their DNA homologies. Three genes, fmrT from the istamycin producer Streptomyces tenjimariensis ATCC 31603, fmrS from the sannamycin producer Streptomyces sannanensis IFO 14239 and fmrH from the sporaricin producer Saccharopolyspora hirsuta ATCC 20501, conferred resistance to FTM-A, kanamycin (Km) and neomycin B (Nm-B), but not to gentamicin (Gm). The other three genes, fmrO from the FTM-A producer Micromonospora olivasterospora ATCC 21819, fmrM from the antibiotic SF-2052 producer Micromonospora sp. SF-2098 (ATCC 31580) and fmrD from the dactimicin producer Dactylosporangium matsuzakiense ATCC 31570, conferred resistance to FTM-A, Km and Gm, but not to Nm-B. No DNA homology was detected between the two types of the resistance genes in Southern-blot analysis. The present results revealed that, in spite of the similarity of their biosynthesis genes, there are at least two different types of resistance genes among the FTM-group antibiotic producers.
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A Macrolide–Lincosamide–Streptogramin B Resistance Determinant from Bacillus anthracis 590: Cloning and Expression of ermJ
More LessThe inducible macrolide-lincosamide-streptogramin B resistance determinant, ermJ, from Bacillus anthracis 590 was cloned in Escherichia coli CSH26. The DNA sequence of ermJ was similar to that of ermK or ermD from B. licheniformis, suggesting that ermK-like genes have been distributed in Bacillus strains by transposition. Expression of ermJ was achieved in a B. subtilis minicell system, and the rRNA methyltransferase product of ermJ was purified. The molecular mass of the enzyme was 58 kDa, and it was concluded to be a homodimer. Its biochemical characteristics were different from those of ermC methyltransferase.
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- Pathogenicity And Medical Microbiology
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Cell-Surface Location of Listeria-Specific Protein p60-Detection of Listeria Cells by Indirect Immunofluorescence
More LessA specific polyclonal antiserum was prepared against a gel-purified 60 kDa extracellular protein of Listeria monocytogenes ATCC 19111 corresponding to protein p60 previously detected in culture broths of L. monocytogenes strains Mackaness and EGD [Kuhn, M. & Goebel, W. (1989), Infection and Immunity 57, 55–61]. Indirect immunogold labelling combined with transmission electron microscopy and high-resolution scanning electron microscopy were used toinvestigate the location and distribution of p60 on the bacterial cell surface. In bacteria grown to the early stationary phase about 25% of the extracellular protein was estimated to be associated with the cell surface. The anti-p60 antiserum proved to be Listeria-specific. In an indirect immunofluorescence test the antiserum reacted with Listeria strains representing all species and different serotypes, except L. seeligeri, L. welshimeri, L. grayi and L. murrayi. No immunological cross-reactions were observed with 27 strains of bacteria from 16 other genera. The value of the anti-p60 antiserum in developing a diagnostic assay for Listeria cells in environmental samples and foods is discussed.
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Similarity in the EDTA-Soluble Antigens of Clostridium Chauvoei and C. Septicum
More LessThe EDTA-soluble antigens were prepared from whole cells of six strains of Clostridium chauvoei and five strains of C. septicum and were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. SDS-PAGE profiles of the 11 strains were nearly identical, although there were slight variations in molecular mass in adjacent bands. In immunoblot analysis with two antisera against C. chauvoei and three against C. septicum, the antigens of all strains tested reacted with all five antisera and there were no differences in reactivities to the same antiserum between homologous and heterologous antigens. In an immunoblot reacted with a single antiserum, band patterns of 10 of the 11 strains were quite similar. After cross-absorption, antisera to both species lost most of their reactivities not only to heterologous antigens but also to homologous antigens. These results indicate that the two species share many common antigens and that there is a marked similarity in the antigenic properties of EDTA-soluble material.
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Calcium- and Mucin-Binding Proteins of Staphylococci
More LessThe association of staphylococci with the mucus gel that overlays the mucosa of the respiratory tract may lead to clearance of cocci or, in certain conditions such as cystic fibrosis (CF), to colonization. In the present study, a quantitative radioassay was used to study the effect of Ca2+, which is elevated in CF sputa, on the adhesion of 3H-labelled Staphylococcus aureus to submaxillary gland mucin immobilized in MaxiSorp 96-well, break-apart modules. Ca2+ significantly enhanced the adhesion of S. aureus (five strains) and Staphylococcus epidermidis (four strains). The reaction was specific because adhesion was not enhanced in the presence of Mg2+, Ca2+ + EGTA (a Ca2+ chelator) or protamine and was not attributable to hydrophobicity of the test strains. Staphylococcal adhesion was significantly (P⩽0·005) blocked in the presence of highly sialated and sulphated reagents, which suggests that Ca2+ binds to the sialic acid and sulphate residues of immobilized mucin. The Ca2+ -binding sites on the surface of S. aureus were trypsin-sensitive; in addition, 125I-labelled solubilized S. aureus surface proteins reacted with immobilized mucin in a direct binding assay, and the reaction was significantly enhanced by Ca2+. Autoradiography demonstrated that 45Ca bound directly to two polypeptides (M r 170000 and 150000) of solubilized staphylococcal surface proteins separated by SDS-PAGE, and that 125I-labelled mucin bound directly to three staphylococcal polypeptides (M r 40000, 35000, and 29000). These results suggest that S. aureus adhesion to mucin is mediated by at least two mechanisms: via Ca2+-binding surface proteins in the presence of Ca2+ and via mucin-binding surface proteins unrelated to Ca2+.
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Fusion of the Genes Encoding Escherichia Coli Heat-Stable Enterotoxin b (STb) and the Maltose-Binding Protein to Obtain Mature STb Enterotoxin
More LessThe heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3–4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.
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Cloning, Characterization and Sequencing of two Haemagglutinin Genes from Eikenella Corrodens
More LessEikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections. From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins. By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct. Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31·5 kDa and two of approximately 14 kDa each. Expression of each of the proteins appears to be under the control of an Eikenella promoter(s). Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa. Expression of both of these proteins in E. coli requires the lac promoter in the vector. By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31·5 kDa polypeptide identified as the haemagglutinin. Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2. The nucleotide sequences of both haemagglutinin genes were determined and are presented. Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins. These are the first genes of this genus to be cloned and sequenced.
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Cloning and Sequencing of Two Type 4 (N-Methylphenylalanine) Pilin Genes from Eikenella Corrodens
More LessEikenella corrodens is a Gram-negative microaerophilic rod which is gaining recognition as an important human pathogen. We have previously reported the cloning and expression in Escherichia coli of a 3·6 kb Eik. corrodens genomic DNA fragment which encodes a 31·5 kDa haemagglutinin. Maxicell analysis revealed that this fragment also encodes two proteins of approximately 14 kDa. Nucleotide sequencing of the 2·2 kb fragment upstream of the haemagglutinin gene revealed two open reading frames with strong homology to genes encoding pilin subunit proteins of the type 4 or N-methylphenylalanine class. The two pilin genes, ecpA and ecpB, are complete and are expressed in E. coli. Southern analysis of ten additional Eik. corrodens strains revealed that all possess fragments homologous to ecpA. These data represent the first molecular evidence for pili in E. corrodens.
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- Physiology And Growth
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Characterization of a Calcium Porter of Streptococcus Pneumoniae Involved In Calcium Regulation of Growth and Competence
More LessIt is shown that Streptococcus pneumoniae possesses a Ca2+ transporter, sensitive to the amiloride derivative 2′,4′-dimethylbenzamil (DMB), which is essential for grown at high Ca2+-concentrations, and which mediates the triggering by Ca2+ of competence for genetic transformation in the exponential phase and autolysis in the late exponential phase. DMB inhibited both Ca2+ transport and the Ca2+ response. Kinetic analysis of 45Ca2+ transport in ATP-depleted S. pneumoniae revealed an electrogenic influx sensitive to DMB. This transport was cooperative with respect to Ca2+ concentration, and exhibited a Hill coefficient (nH) of 2. In bacteria pre-loaded with 45Ca2+, a DMB-sensitive efflux could be triggered by an imposed Na+ gradient. The efflux kinetics showed the same cooperativity profile as Ca2+ concentration and a similar nH value to that of influx, suggesting a possible Na+/Ca2+ antiport. Cooperativity of transport was lowered (nH = 1) by a mutation that confers resistance to DMB and abolishes the Ca2+ response. These results demonstrate that DMB-sensitive Ca2+ transport is essential for growth and competence regulation. The role of the DMB-sensitive porter involved in Ca2+ circulation and in Ca2+ homeostasis and its possible regulation by competence factor are discussed.
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Oxygen-Dependent Alginate Synthesis and Enzymes in Pseudomonas Aeruginosa
More LessAlginate production by the highly alginate-producing Pseudomonas aeruginosa 8821M was maximal at a dissolved oxygen tension (DOT) of 5% of air saturation. Lower DOT limited growth and alginate synthesis. At higher DOT values up to 70% of air saturation, the specific alginate production rate decreased. Nevertheless, the molecular mass of the alginate increased at higher aerations, as indicated by the viscosity of solutions of the isolated biopolymer. The specific activity of the four enzymes leading to GDP-mannuronic acid formation, phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP) and GDP-mannose dehydrogenase (GMD), increased with DOT of up to 25%. At higher DOT, however, only GMP and GMD maintained their maximum values. Changes observed at high oxygen concentrations in the relative activities of PMI and GMP, which are activities of the same bifunctional protein, were attributed to the much higher sensitivity of PMI activity to irreversible oxidative inactivation. The less pronounced decrease of PMM activity at high DOT correlated with an intermediate sensitivity to oxidative inactivation, but could also be related to sequential induction of PMM by the product of the PMI reaction. Thus, oxygen-dependence of alginate synthesis was at least partially the effect of DOT on GDP-mannuronic acid formation. Optimal aerations for maximal alginate production (DOT = 5–10%) were below the aeration level (70%) that led to the highest viscosity. These results suggest that, like GMD, polymerization activity is not very sensitive to oxidative inactivation and they are consistent with the hypothesis that polymerization is dependent on GMD activity, or is regulated in a similar way.
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The role of Polyamine Metabolism in Dimorphism of Yarrowia Lipolytica
More LessWe have devised a convenient procedure to induce the yeast-to-mycelium transition of Yarrowia lipolytica in conditions which avoid the occurrence of the reverse process during the period of study. Yeast cells in late exponential phase were resuspended in water and cooled down to 4 °C for at least 15 min, then heat-shocked by inoculation into a pre-warmed (30 °C) medium containing N-acetyl-d-glucosamine. Under these conditions, yeast cells developed into large branching filaments which continued elongating for more than 24 h. Further, ornithine decarboxylase (ODC) activity and polyamine cell pools increased compared to those of cells maintained in glucose medium, which continued yeast-like growth. Addition of ODC inhibitors blocked mycelial development, but only if added during a critical initial period after which they had no effect. At effective concentrations, ODC inhibitors had no significant effect on cell growth. Comparative studies of intact and permeabilized cells suggest that this selective effect is probably due to the location of ODC in more than one cell compartment, one of them being inaccessible to the drugs. Blocking of the morphological transition by ODC inhibitors was specifically reversed by putrescine, and by growing the cells in the presence of 5-azacytidine. It is suggested that the effect of the latter compound is related to its capacity to inhibit DNA methylation, indicating a relationship between polyamines and DNA methylation at the onset of the differentiation process.
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Saccharomyces Cerevisiae has an Inducible Response to Menadione which differs from that to Hydrogen Peroxide
More LessExponential phase cells of Saccharomyces cerevisiae treated with the superoxide free-radical generating agent menadione (MD; 0·2 mm) for 60 min adapted to become resistant to the lethal effects of a higher concentration of MD (4 mm). Inhibition of protein synthesis by treatment with cycloheximide totally prevented the adaptation to MD, indicating that this is an inducible response completely dependent on protein synthesis; this differs from the situation with peroxide in which only some of the adaptive response is cycloheximide-sensitive. Cells subjected to heat shock (23 to 37 °C) or treatment with hydrogen peroxide (H2O2; 0·2 mm, 60 min) became more resistant to 4 mm-MD; however, MD pretreatment did not induce any thermotolerance or resistance to peroxide. These differences between the response to MD and H2O2 were reflected in the results of l-[35S]methionine labelling studies. Using one-dimensional electrophoresis, only one polypeptide (60 kDa) was seen to be induced by 0·2 mM–MD and this was also induced by heat shock but not peroxide stress. With heat shock or peroxide treatment the induction of at least 10 polypeptides was detected using this approach. Using an isogenic petite strain, it was found that functional mitochondria were needed for conferring full resistance to MD, but that induction of the adaptive response was not dependent on mitochondrial function.
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- Plant-Microbe Interactions
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Evaluation of a Strategy for Identifying Nodulation Competitiveness Genes in Rhizobium Leguminosarum Biovar Phaseoli
More LessSummary: Rhizobium leguminosarum biovar phaseoli strain KIM5s is consistently much more competitive than strain CE3 in nodulation of beans (Phaseolus vulgaris L.) in the laboratory and in the field. To identify genes that contribute to the competitiveness of KIM5s, we transferred a cosmid library containing KIM5s DNA into CE3 and applied the transconjugants to bean plants to allow the plants to enrich for those with enhanced nodulation competitiveness. The nodule isolates were then applied to plants for further enrichment. Of 75 isolates from nodules sampled after the two enrichments, 9 were more competitive than CE3. For example, when outnumbered in the inocula 40-fold by a reference strain, these nine strains typically occupied 15–40% of the nodules compared with 0–3% for CE3. However, when these strains were cured of the cosmids, they remained highly competitive, demonstrating that the enhanced competitiveness of the strains was not associated with the cosmids. We found no evidence for cosmid insertion into the chromosome or for cosmid-induced genetic changes in these cured strains. We found some evidence suggesting that their altered competitiveness was due to spontaneous genetic changes that did not involve the cosmids. Although these highly competitive variants remain genetically uncharacterized, they may provide insight into bacterial traits that contribute to, or detract from, successful nodulation competitiveness.
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- Systematics
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Identification and Classification of Lactobacillus Acidophilus, L. Gasseri and L. Johnsonii Strains by SDS-PAGE and rRNA-Targeted Oligonucleotide Probe Hybridization
Thirty-two strains originally identified as Lactobacillus acidophilus and L. gasseri were screened for their taxonomic homogeneity by SDS-PAGE of whole-cell proteins. After numerical comparison of the resulting protein electrophoretic fingerprints, two well-delineated clusters were detected. The majority of the strains grouped in one electrophoretic cluster, which contained the type strain of L. acidophilus and corresponds to DNA group A1 of Johnson, J. L., Phelps, C. F., Cummins, C. S., London, J. & Gasser, F. (1980; International Journal of Systematic Bacteriology 30, 53–68). Another cluster corresponded to DNA group B. It contained two subclusters, which agreed perfectly with DNA subgroups B1 (L. gasseri) and B2 (L. johnsonii), respectively. The 23S rRNA genes were partially sequenced and 23S-rRNA-targeted oligonucleotide probes were designed for identification of DNA groups A1, B1 and B2. Probe Lbg reacted with all strains of electrophoretic cluster B1 (L. gasseri), probe Lbj hybridized with strains of cluster B2 (L. johnsonii) and probe Lba with strains of cluster A1 (authentic L. acidophilus). The probes were successfully used for the identification of strains belonging to the respective species. The phylogenetic relationship of a representative of L. johnsonii was determined by comparative sequence analysis of the 16S rRNA genes. It is very closely related to L. gasseri.
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Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA
More LessA method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.
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