- Volume 133, Issue 8, 1987
Volume 133, Issue 8, 1987
- Biochemistry
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The Agarase Gene (dag A) of Streptomyces coelicolor A3(2): Affinity Purification and Characterization of the Cloned Gene Product
More LessSUMMARY: The coding and regulatory sequences of the agarase gene of Streptomyces coelicolor A3(2) were cloned in Streptomyces lividans 66 on the plasmid vector pIJ61, resulting in a several hundredfold increase in the production of the secreted protein. Subcloning experiments localized the sequences required for agarase production and for the mediation of carbon catabolite repression to a segment of about 1·2 kb. A simple protein purification procedure that uses affinity binding of agarase to agarose beads was developed. Preliminary characterization of the enzyme, together with the results of in vitro transcription-translation studies, suggest that the intracellular form of agarase (about 34 kDa) possesses a signal sequence that is cleaved upon secretion across the cell membrane to produce an extracellular protein of about 29 kDa.
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A Mutant of Candida albicans Deficient in β-N-Acetylglucosaminidase (Chitobiase)
More LessSUMMARY: A mutant of Candida albicans ATCC 10261 was isolated that was defective in the production of β-N-acetylglucosaminidase (chitobiase). The mutant grew normally in minimal medium supplemented with either glucose or N-acetyl-D-glucosamine (GlcNAc) as carbon and energy source, and the cells formed germ-tubes at 37°C when induced to do so with GlcNAc. However, unlike the wild-type parent strain, the mutant strain did not utilize N, N'-diacetylchitobiose for growth. The mutant and parent strains had similar growth rates on glucose or GlcNAc, similar rates of uptake of these sugars and similar rates of 14C-labelled amino acid incorporation. The chitobiase mutant did, however, contain 53-85% more chitin than the wild-type strain. No reversion of the mutant phenotype was observed following induction of mitotic recombination with UV light, suggesting that the mutant allele (chi) was carried homozygously in the chitobiase-deficient mutant. Although the chitobiase-deficient mutant was pathogenic, it was not as virulent as the wild-type strain.
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Iron Transport in Mycobacterium smegmatis: Occurrence of Iron-regulated Envelope Proteins as Potential Receptors for Iron Uptake
More LessSUMMARY: Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0·02 μg Fe ml-1) and iron-sufficient (2·0 to 4·0 μg Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins; the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.
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Purification and Characterization of Chloramphenicol Acetyltransferase from Flavobacterium CB60
More LessSUMMARY: From the highly chloramphenicol-resistant cytophaga-like bacterium Flavobacterium CB60, which can both acetylate chloramphenicol and degrade it in co-metabolism, the chloramphenicol acetyltransferase (CAT) was purified to homogeneity and characterized. The purification included fractional precipitation with ammonium sulphate and two affinity chromatography steps, eluting CAT the first time with 5 mM-chloramphenicol and the second time with a linear gradient (0–10 mM) of chloramphenicol. The purification was 3979-fold. Properties of this CAT were investigated and compared with CATs from other bacteria. Although CAT from Flavobacterium CB60 shares some properties with the enzymes from Escherichia coli and other Gram-negative bacteria - especially with CATII and CATIII - it has distinct properties like extreme heat lability and the inability to produce diacetylchloramphenicol, so that it might be regarded as a new variant.
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A Xanthan-gum-like Polysaccharide from Acetobacter xylinum
More LessSUMMARY: A new exopolysaccharide, secreted in addition to cellulose, has been isolated from the culture medium of Acetobacter xylinum NRRL B42. This polysaccharide, for which the name acetan is proposed, contains glucose, mannose, glucuronic acid and rhamnose in a molar ratio of 4:1:1:1. On the basis of methylation, thin-layer, paper and gas-liquid chromatography, paper electrophoresis and mass spectrometry studies of the degradation products obtained by total and partial hydrolysis and acetolysis of acetan, the following structure is proposed for its repeating unit:
Since our previous work with this strain demonstrated the in vitro synthesis of a lipid-linked heptasaccharide with the same structure, the possibility of acetan being the result of its polymerization is discussed. One to two O-acetyl residues per repeating unit are also present in positions not yet determined.
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Hydroquinone as the Ring-fission Substrate in the Catabolism of 4-Ethylphenol and 4-Hydroxyacetophenone by Pseudomonas putida JD1
More LessSUMMARY: A bacterium capable of growth on 4-ethylphenol was isolated from soil and identified as Pseudomonas putida. Intact cells grown on 4-ethylphenol rapidly oxidized 4-hydroxyaceto-phenone as well as growth substrate and the bacterium was also capable of growth on 4-hydroxyacetophenone. The initial enzymes for 4-ethylphenol catabolism were still present, although at lower activities, in succinate-grown cells which oxidized 4-ethylphenol to 4-hydroxyacetophenone. Extracts of 4-ethylphenol-grown cells oxidized 4-hydroxyacetophenone when provided with NADPH. When this activity was partially purified a stoichiometry of 1 μmol O2 consumed per μmol of substrate was observed with the production of hydroquinone as required for a monooxygenase producing 4-hydroxyphernyl acetate followed by hydrolysis by an esterase. Cell extracts contained esterase activity and hydrolysed 4-hydroxyphenyl acetate to yield hydroquinone. Intact cells converted the analogue, acetophenone, into phenol. Hydroquinone served as the ring-fission substrate and was cleaved by an O2-requiring reaction. The enzymes of the proposed pathway were induced by growth on 4-ethylphenol or 4-hydroxyacetophenone.
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Enzymological Features of Aromatic Amino Acid Biosynthesis Reflect the Phylogeny of Mycoplasmas
More LessSUMMARY: Acholeplasma laidlawii possesses a biochemical pathway for tyrosine and phenylalanine biosynthesis, while Mycoplasma iowae and Mycoplasma gallinarum do not. The detection of 7-phospho-2-dehydro-3-deoxy-d-arabino-heptonate (DAHP) synthase (EC 4.1.2.15), dehydro-shikimate reductase (EC 1.1.1.25) and 3-enol-pyruvoylshikimate-5-phosphate synthase (EC 2.5.1.19) activities in cell-free extracts established the presence in A. laidlawii of a functional shikimate pathway. l-Phenylalanine synthesis occurs solely through the phenylpyruvate route via prephenate dehydratase (EC 4.2.1.51), no arogenate dehydratase activity being found. Although arogenate dehydrogenase was detected, l-tyrosine synthesis appears to occur mainly through the 4-hydroxyphenylpyruvate route, via prephenate dehydrogenase (EC 1.3.1.12), which utilized NAD+ as a preferred coenzyme substrate. l-Tyrosine was found to be the key regulatory molecule governing aromatic biosynthesis. DAHP synthase was feedback inhibited by l-tyrosine, but not by l-phenylalanine or l-tryptophan; l-tyrosine was a potent feedback inhibitor of prephenate dehydrogenase and an allosteric activator of prephenate dehydratase. Chorismate mutase (EC 5.4.99.5) was sensitive to product inhibition by prephenate. Prephenate dehydratase was feedback inhibited by l-phenylalanine. It was also activated by hydrophobic amino acids (l-valine, l-isoleucine and l-methionine), similar to results previously found in a number of other genera that share the Gram-positive line of phylogenetic descent. Aromatic-pathway-encoded cistrons present in saprophytic large-genome mycoplasmas may have been eliminated in the parasitic small-genome mycoplasmas.
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Purification and Properties of the Endo-1,4-β-glucanase from Bacillus subtilis
More LessSUMMARY: Carboxymethylcellulase (endo-1,4-β-glucanase; EC 3.2.1.4) was purified from the culture filtrate of Bacillus subtilis AU-1 by (NH4)2SO4 precipitation, Avicel affinity chromatography, DEAE Sephadex G-75 chromatography and Sulphopropyl Sephadex C-50 chromatography. The enzyme was purified 36-fold and had an M r of 23000 as determined by gel filtration on a Sephadex G-75 column. The pH optimum of the purified enzyme was 5·5; the enzyme was stable at 65°C. Activity of the purified enzyme was significantly reduced by Cu2+, Pb2+, Sn2+, Ag+, Hg2+ and Fe2+, but was increased by 139·5% in the presence of Co2+. Inhibition studies indicated that the purified enzyme was either a metalloprotein or required certain metal ions for activation/stabilization; that iron was not a prosthetic group of the enzyme; that a tryptophanyl group was not involved in enzyme action; and that reduced thiol groups were required for enzyme activity and involved in the active site of the enzyme. The K m of the purified enzyme for carboxymethylcellulose was 4 mg ml-1, and the V max for carboxymethylcellulose hydrolysis was 0·42 mg d-glucose min-1 (mg protein)-1.
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Aerobic and Anaerobic Uptake of Sugars in Schizosaccharomyces pombe
More LessSUMMARY: Aerobic cells of the yeast Schizosaccharomyces pombe accumulated 2-deoxy-d-glucose (2-DOG) and glucosamine, but were unable to accumulate 3-O-methyl-d-glucose, 6-deoxy-d-glucose, d-xylose and d-arabinose. Uptake of all sugars tested displayed saturation kinetics; however, only 2-DOG, glucosamine and d-glucose exhibited mutual competitive inhibition of uptake and the phenomenon of exchange transport. Thus, they share a common ‘glucose transport system’. Uncouplers inhibited sugar accumulation or induced sugar outflow from preloaded cells. The uptake of sugars of the glucose transport system was pH dependent and was accompanied by a stoicheiometric cotransport of H+. Since millimolar concentrations of the lipophilic cation tetraphenylphosphonium (which depolarizes the membrane potential) prevented sugar accumulation, the transport was electrogenic. Thus, the glucose carrier is a H+-symporter. Accumulation of sugars was inhibited by the plasma membrane ATPase inhibitors Dio-9 and N, N’-dicyclohexylcarbodiimide. Anaerobic cells of S. pombe were virtually unable to transport 2-DOG and glucosamine. However, when energized by glucose, the accumulation of both sugars was completely restored. This anaerobic transport was also catalysed by the glucose carrier.
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- Development And Structure
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Hexagonal Periodicity in the Outer Membrane of Bacteroides buccae
More LessSUMMARY: In Bacteroides buccae, a hexagonally arranged periodic structure was found in the outer membrane (OM), in addition to hexagonal lattices present in its external surface layer (S-layer). This crystalline OM protein (COMP) was present as patches on the concave fracture face (the outer leaflet) of the OM in freeze-fractured cells. Occasionally, hexagonally arranged structures could also be seen on the convex fracture face of the OM as ‘fingerprints’ of the COMP. The OM proteins were isolated and analysed by gel electrophoresis. The major band protein had an apparent molecular mass of 17 kDa. Whether the minor band proteins are also components in the structure of the COMP remains to be elucidated. Other oral Gram-negative anaerobic rods studied did not show any periodicity in their OM.
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- Ecology
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The Detection of Starvation-specific Antigens in Two Marine Bacteria
More LessSUMMARY: Antisera produced against starved cells of two marine bacteria (a Vibrio sp. and an unidentified Gram-negative motile rod) were titrated in order to quantify with respect to time of starvation the appearance of starvation-specific antigens. Polyacrylamide gels of lipopolysaccharide digests, and of total protein and membrane and periplasmic fractions, were prepared, blotted and immunoassayed to determine the location(s) of the antigenic response. For the Vibrio isolate, no starvation-specific antigens were detected, but such antigens were detected for the other isolate; they were proteinaceous and were located in the outer membrane and periplasmic space. Titrations of whole cells indicated that the antigenic change in the cell surface occurred in the initial phase of starvation and increased during the first 14 h of the starvation period studied.
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- Genetics And Molecular Biology
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The R46 Site-specific Recombination System is a Homologue of the Tn3 and γδ (Tn1000) Cointegrate Resolution System
More LessSUMMARY: The nucleotide sequence of the R46 site-specific recombination system has been determined. The organization of the recombination gene (per R46) and the site at which it acts (per site), together with the extensive sequence homology displayed with the tnpR genes and res sites of the transposons Tn3 and γδ (Tn1000), suggests that they have been derived from a Tn3-like element. These site-specific recombination functions of R46 play a role in plasmid maintenance.
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The Amidase Regulatory Gene (amiR) of Pseudomonas aeruginosa
More LessSUMMARY: Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing sub-cloned DNA fragments, the amiR gene was located within a l kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.
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A Rapid and Efficient Method for Plasmid Transformation of Klebsiella pneumoniae and Escherichia coli
More LessSUMMARY: A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5al and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5al than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5al and E. coli K12.
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Plasmid Transformation of Azotobacter vinelandii OP
More LessSUMMARY: Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron-and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif +). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42°C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HindII -digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.
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pTDN1, A Catabolic Plasmid Involved in Aromatic Amine Catabolism in Pseudomonas putida mt-2
More LessSUMMARY: The ability of Pseudomonas putida mt-2 strain UCC2 to grow with aniline, m- and p-toluidine and m-toluate (the Tdn+ phenotype) is plasmid encoded. Strain UCC2 contains two plasmids of which pUCC2 is a deleted derivative of the TOL plasmid pWW0 (Worsey & Williams, 1975) and can be lost from the strain with no effect on the Tdn+ phenotype. The second plasmid in strain UCC2, pTDN1, harbours genes involved in the Tdn+ phenotype and is conjugative. The catechol 2,3-dioxygenase (C230) structural gene resident on pTDN1 has been cloned into the broad host range vector pKT231. The relative specific activity towards substituted catechols of C23O expressed in the cloned fragment in Escherichia coli is similar to that expressed in P. putida strain UCC2.
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Analysis of the Inhibition of Sporulation of Bacillus subtilis Caused by Increasing the Number of Copies of the spO0F Gene
More LessSUMMARY: The Bacillus subtilis spoOF gene was cloned on a 6·3 kbp BglII fragment. The effect on sporulation of amplification of the spoOF region was examined. Sporulation was inhibited to less than 5% of that of the parental strain when as few as four copies of the spoOF region were present. Subclones, constructed in autonomous or integrative vectors, were used to demonstrate that the region responsible for the copy-number-dependent asporogeny corresponded closely with the spoOF structural gene. A possible mechanism for this effect is discussed.
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The Major Acid-soluble Proteins of Bacillus subtilis Spores: Partial Amino Acid Sequence and Forespore Location of Their mRNAs
More LessSUMMARY: In Bacillus subtilis the α, β, γ and δ components comprise 80–90% of the total acid-soluble spore proteins (ASSPs). Sequence analysis demonstrates that α and β share 32 of their first 36 amino acids and are closely related to the A and C ASSPs of Bacillus megaterium spores, confirming the results of analysis of their cloned genes. Despite the difference in apparent size of γ and δ, they have identical N-terminal sequences (37 residues). Unless γ and δ derive from very recently duplicated genes, it appears that γ is derived from δ, either in vivo or during isolation. Although the sequenced regions of γ and δ have no homology to α and β, outside of the previously recognized pentapeptide recognition sequence for the spore endopeptidase, they share 10 and 15 residue peptides flanking this sequence with ASSP B of B. megaterium, but in reverse order. At least two groups of ASSPs have, therefore, been conserved between B. subtilis and B. megaterium: the multigene ACαβ family and the Bγ(δ) group. Sequence conservation in each group implies selection for functions in addition-to storage. Both the α and β components of B. subtilis ASSPs and their mRNAs are located in the forespore compartment of cells at t 5·5 of sporulation, the time of most rapid ASSP synthesis. The sizes of these transcripts (250–350 bp) and their ability to direct the in vitro synthesis of ASSPs of mature size, indicate that genes for these ASSPs are monocistronic, consistent with dispersed map location. Synthesis of ASSPs is, therefore, coordinately controlled by selective transcription in the forespore.
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Narrow-host-range IncP Plasmid pHH502-1 Lacks a Complete IncP Replication System
More LessSUMMARY: Plasmid pHH502-1 shows incompatibility only towards members of the IncP group, but has a narrower host range than typical members of that group. In contrast to other IncP plasmids its replication was not affected by a high-copy-number plasmid carrying the replication origin (oriV) of IncP plasmid RK2. Southern blotting of pHH502-1 revealed homology to oriV, consistent with its incompatibility phenotype, but no homology to trfA, the essential replication gene of RK2. Thus it is probable that pHH502-1 does not possess a functional IncP replication system, accounting for its restricted host range. A restriction map of pHH502-1 was constructed and the mercury-resistance determinant was localized to Tn735, which also carries the trimethoprim-resistance determinant and is related to Tn21. The presence of a korB-like function on pHH502-1 was also demonstrated.
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Characterization of Three Group A Klebicin Plasmids: Localization of Their E Colicin Immunity Genes
More LessSUMMARY: We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin Al-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin Al-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.
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Characterization of a Family of Temperate Actinophages of Faenia rectivirgula
More LessSUMMARY: Six temperate phages of the thermophilic actinomycete Faenia rectivirgula were characterized by restriction analysis and found to be closely related: (1) three phages (øFR113, øFR371, øFFR114) isolated from lysogenic strains, (2) a deletion-derivative of øFR113 (øFR755R) and (3) two phages (øFR747 and øFRG9) isolated from soil. øFR371 differs from øFR113 by a deletion not overlapping with that in øFR755R. The restriction maps of øFR114 and øFR113 are very similar. øFR747 and øFRG9 belong to the same family as shown by the comparison of their restriction fragment patterns.
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Molecular Cloning of Streptococcus bovis Lactose Catabolic Genes
More LessSUMMARY: The hydrolysis of lactose in Streptococcus bovis was shown to be catalysed by β-D-galactosidase. To isolate lactose catabolic genes, a gene library of S. bovis DNA was constructed in bacteriophage λ47.1 and recombinants expressing β-D-galactosidase activity were detected using 5-bromo-4-chloro-indoyl-β-D-galactoside (X-Gal). The gene was cloned on a 7·8 kb DNA restriction fragment into pBR322 to generate the recombinant plasmid pHG1, which also encoded S. bovis lactose permease, thiogalactoside transacetylase and lactose repressor protein. The position and orientation of the four genes were determined by subcloning and transposon mutagenesis. The β-D-galactosidase, lactose permease and thiogalactoside transacetylase genes constitute an operon controlled by the repressor protein. β-D-Galactosides induced expression of the S. bovis lac genes in Escherichia coli while glucose had no effect. The nucleotide sequence of the presumptive regulatory region of the S. bovis lac operon was determined and compared with the corresponding E. coli sequence. The operators and the 5’ end of the lacZ genes showed strong identity. The catabolite activator protein binding sequence, present in the E. coli promoter, was absent from the corresponding S. bovis region.
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Cloning, Expression and Release of a Vibrio alginolyticus SDS-resistant Ca2+-dependent Exoprotease in Escherichia coli
More LessSUMMARY: A Vibrio alginolyticus SDS-resistant, Ca2+-dependent serine exoprotease gene, cloned on a 7-1 kb DNA fragment in the recombinant plasmid pVP100, was expressed from its own promoter in Escherichia coli. Although active exoprotease was produced by late stationary phase E. coli(pVP100) cultures after 15 h incubation in proteinaceous medium containing Ca2+, transcription and translation of the exoprotease occurred before 6 h, during exponential growth. The cloned exoprotease was synthesized as a pool of inactive precursor molecules during exponential growth, and released as active exoprotease 8 h later by a process which did not require protein synthesis or involve cell lysis. Release of the exoprotease by E. coli(pVP100) was not inhibited by o-phenanthroline, quinacrine or cerulenin. Supernatant samples from E. coli(pVP100) cultures contained two SDS-resistant exoproteases with apparent M r values of approximately 54000 and 39000. The cloned exoprotease activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes.
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Characterization of the Pseudomonas aeruginosa Alginate (alg) Gene Region II
More LessSUMMARY: Pseudomonas aeruginosa region II alginate genes are involved in the biosynthesis of the uronic acid containing exopolysaccharide, alginic acid. We have subcloned and overexpressed various DNA fragments contained within region II in an attempt to further characterize and more precisely localize the genes involved in alginate production. Overexpression of the genes controlling alginate biosynthesis within region II was accomplished by placing various cloned restriction fragments under the transcriptional control of the hybrid trp-lac (tac) promoter, and plasmid encoded proteins were examined in a maxicell expression system. We correlated various region II plasmid constructions with the ability to complement specific alginate negative (alg) mutants and code for polypeptides. Several proteins suspected of being involved in alginate production were encoded by sequences within region II. The results of this study further reveal that the transcriptional orientation of the alg loci within region II appears to be in the direction from argF to pmi. The specific activities of phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), two enzymes involved in the formation of the alginate precursor GDP-mannuronic acid, were measured in region II alg mutants and in cells overexpressing cloned segments from region II. Based on these enzyme measurements, we conclude that the remaining region II alg genes do not encode either PMM or GMP. These results support the suggestion that the remaining alg genes in region II are likely to be involved in post GDP-mannuronic acid processing events such as mannuronic acid transport, polymerization, secretion, epimerization and acetylation.
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- Pathogenicity And Medical Microbiology
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Haemagglutination and Tissue Culture Adhesion of Gardnerella vaginalis
More LessSUMMARY: Six strains of Gardnerella vaginalis were studied to examine the adhesin-receptor mechanism involved in their attachment to human red blood cells and an epithelial tissue culture cell line (McCoy). The adhesins involved in the attachment of the bacteria to each of these cells were proteinaceous but showed marked differences after various chemical or physical treatments, indicating that separate adhesins were present. Haemagglutinating strains were more hydrophobic than tissue-culture-adherent strains. Haemagglutination of human red blood cells by strains of G. vaginalis was inhibited by galactose, lactose, N-acetylneuraminic acid and phosphatidylserine. In contrast, the tissue-culture adherence of strains was not inhibited by these substances.
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Susceptibility of Micro-organisms to Active Oxygen Species: Sensitivity to the Xanthine-oxidase-mediated Antimicrobial System
More LessSUMMARY: Xanthine oxidase with acetaldehyde as substrate (the XOA system) generated superoxide anion and hydrogen peroxide, but this system had only weak bactericidal activity. Addition of Fe2+ and EDTA to the XOA system (XOA-Fe-EDTA system) increased bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Salmonella typhimurium, although both Mycobacterium tuberculosis and Candida albicans remained highly resistant. Catalase (H2O2 scavenger) and mannitol (OH scavenger) almost completely inhibited the bactericidal activity of the XOA-Fe-EDTA system whereas SOD (O2 - scavenger) was less inhibitory, Azide (1O2 scavenger) caused no such inhibition. The results suggest the possible role of OH, H2O2 and O2 - in the XOA-Fe-EDTA-mediated antimicrobial system, as effector molecules. There was no correlation between resistance of a given bacterium to active oxygen and the level of endogenous active oxygen-scavengers.
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Relationship between the Susceptibility of Various Bacteria to Active Oxygen Species and to Intracellular Killing by Macrophages
More LessSUMMARY: The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 ≥ Listeria monocytogenes EGD ≥ Salmonella typhimurium HKB-1 ≥ Staphylococcus aureus Smith ≥ Mycobacterium tuberculosis H37Rv ≥ Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (Møs), C. albicans, Staph, aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal Møs (as measured by chemiluminescence), Staph, aureus showed the highest activity followed by the other organisms in the following order: C. albicans > E. coli > L. monocytogenes ⋍ M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph, aureus and E. coli to Mø bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.
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Amino Acid Sequence and Antigenicity of the Amino-terminus of the 168 kDa Adherence Protein of Mycoplasma pneumoniae
More LessSUMMARY: The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits. Antibodies against this peptide epitope could be demonstrated in sera taken during natural M. pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its homologous antibody failed to inhibit adherence of M. pneumoniae.
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Flanking and Internal Regions of Chromosomal Genes Mediating Aerobactin Iron Uptake Systems in Enteroinvasive Escherichia coli and Shigella flexneri
More LessSUMMARY: We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pCoIV-K 30-encoded ferric aerobactin receptor.
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Monoclonal Antibodies against the Haemolysin of Vibrio vulnificus
More LessSUMMARY: The extracellular haemolysin produced by Vibrio vulnificus strain FCC was partially purified from the culture supernate by sequential ammonium sulphate precipitation, gel filtration with Sepharose 4B, and DEAE-Sephacel ion-exchange column chromatography. Using this semi-purified haemolysin as the antigen, several monoclonal antibodies (MAbs) were established; they were all of the IgG2b class with lambda light chains. One representative MAb, 6F8D, completely neutralized the haemolytic activity and mouse lethal activity of extracellular toxin(s). In immunoblotting analysis of the peptides of the semi-purified haemolysin separated by SDS-PAGE, this MAb reacted, in particular, with a 36 kDa peptide. These findings suggest that the haemolysin is probably identical to the lethal toxin in the culture supernate of V. vulnificus strain FCC, which contained the 36 kDa peptide.
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- Physiology And Growth
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Degradation of Aflatoxin by Aspergillus flavus
More LessSUMMARY: Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.
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Sulphur Dioxide Resistance in Saccharomyces cerevisiae and Saccharomycodes ludwigii
More LessSUMMARY: Saccharomyces cerevisiae was unable to grow in media containing above about 1·5 mm free sulphite at pH 4·0, whereas Saccharomycodes ludwigii grew at the same pH value in the presence of 7·8 mm free sulphite. Expressed in terms of μl of intracellular water, the initial velocity of sulphite accumulation by S'codes ludwigii was approximately twice that of S. cerevisiae, although the former yeast accumulated at equilibrium only about one-third of the amount of sulphite accumulated by S. cerevisiae. A Woolf-Hofstee plot for accumulation of SO2 by S'codes ludwigii at pH 3·0 and 30°C gave a vertical line. Incorporation of sulphite in growth media induced excretion of acetaldehyde by both yeasts, the rate being greater by S'codes ludwigii than S. cerevisiae. Acetaldehyde excretion was accompanied by release of lower concentrations of pyruvate. Excretion of 2-oxoglutarate was barely detectable. It is suggested that the greater resistance of S'codes ludwigii to sulphite, compared with S. cerevisiae, may be explained partly by its decreased capacity to accumulate the compound, and partly by its ability to produce more acetaldehyde.
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Production of Long-chain Alcohols by Yeasts
More LessSUMMARY: Fourteen yeast strains from six genera were analysed for the presence of long-chain alcohols. Six strains from three genera contained long-chain alcohols, highest levels being found in Candida albicans. The alcohols were identified and determined by TLC, GLC and GLC-MS. The major long-chain alcohols synthesized by these organisms were saturated, primary alcohols with C14, C16 or C18 chain length. Unsaturated long-chain alcohols were not detected. In all strains that produced long-chain alcohols, the relative proportions were C16 > C18 > C14. Long-chain alcohol contents were higher in organisms from anaerobically, as compared with aerobically, grown cultures reaching about 650 μg (g dry wt organisms)-1 in stationary-phase cultures of C. albicans. In cultures of C. albicans, synthesis of long-chain alcohols occurred only after the end of exponential growth. The alcohols were predominantly present as free alcohols. The fatty-acyl chain-length profile of the triacylglycerol and to a lesser extent the sterol/wax ester fractions from C. albicans reflected that of the long-chain alcohols produced by this yeast.
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Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: Intracellular pH and the Effect of Membrane Depolarizing Compounds
SUMMARY: Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 3P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP. azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.
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Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: the Glucose-induced cAMP Signal Is Not Mediated by a Transient Drop in the Intracellular pH
SUMMARY: Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1–2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31 P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.
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Regulation of Sorbitol Metabolism by Glucose in Clostridium pasteurianum: a Role for Inducer Exclusion
More LessSUMMARY: Cells of Clostridium pasteurianum grown on glucose lacked enzyme systems specific for sorbitol metabolism: the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PTS) and sorbitol-6-phosphate dehydrogenase. These activities were induced by growth on sorbitol, but were partially repressed by glucose. During growth on excess glucose and sorbitol, the sorbitol PTS was absent until the growth rate declined at the onset of stationary phase. Synthesis of at least two PTS proteins, one soluble and one membrane-bound, was repressed in exponentially growing cells. In contrast to the PTS, sorbitol-6-phosphate dehydrogenase was present during the exponential phase.
Glucose and methyl α-glucoside inhibited the activity of the sorbitol PTS in permeabilized cells, and this inhibition was competitive at the level of PEP. This implies that in intact cells sorbitol is excluded by glucose, at least in part due to competition between the glucose and sorbitol phosphotransferases for a common pool of phosphate and energy. Exclusion of sorbitol may be an important factor in the repression of sorbitol metabolism by glucose.
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Formation of a New Cell Wall by Protoplasts of Candida albicans: Effect of Papulacandin B, Tunicamycin and Nikkomycin
More LessSUMMARY: Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.
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Phosphatase Activities in the Life Cycle of Myxococcus coralloides D
More LessSUMMARY: The effect of phosphate on the different stages of the life cycle of Myxococcus coralloides D has been examined. A high concentration of phosphate inhibited the process of fructification, but glycerol-induced myxospore formation and germination were independent of phosphate concentration. Acid and alkaline phosphatases were detected and both were released into liquid medium during the exponential growth phase. On solid medium, phosphatase activities showed different patterns according to the stage of the life cycle. The increase of these two activities was transient during glycerol-induced myxospore formation. Differences in phosphatase activities during germination of glycerol-induced myxospores and fruiting-body myxospores were found.
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Functional Analysis of Ammonium Assimilation Enzymes in Neurospora crassa
More LessSUMMARY: In order to explore the function of each of the four enzymes involved in ammonium assimilation in Neurospora crassa [NADP-dependent glutamate dehydrogenase (GDH), NADH-dependent glutamate synthase (GOGAT) and the α and β isozymes of glutamine synthetase (GS)] growth curves, enzymic activities and intracellular pools of ammonium, glutamate and glutamine were determined in the wild-type as well as in mutants lacking one or two of these biosynthetic enzymes. These parameters were measured in ammonium-limited chemostat-type cultures, in which conditions resembling a continuous culture were achieved for the first time for a filamentous fungus, and in ammonium-excess batch cultures. NADP-dependent GDH appeared to be the main provider of cellular glutamate in the presence of limited and excess ammonium but could be partially replaced by NADH-dependent GOGAT depending upon the presence of GSβ. GOGAT appeared to have the main role in the recycling of glutamine to glutamate, particularly in ammonium-limited conditions. GSβ was the main provider of cellular glutamine but GSα could substitute for this activity if GDH was also present. In conditions of ammonium excess GDH and GOGAT were repressed by glutamate and GS was repressed by glutamine, as previously found. However, in conditions of ammonium limitation these enzymes appeared to escape nitrogen repression.
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Vacuoles Play a Decisive Role in Calcium Homeostasis in Neurospora crassa
More LessSUMMARY: Mutants of the filamentous fungus Neurospora crassa which grow well only in a medium in which the concentration of calcium is lower than in the medium used for culture of the wild-type strain were isolated by use of the ionophore A23187 and medium which contained a high concentration of calcium. These mutants showed a significantly lower uptake of calcium into their vacuoles than the wild-type, while other activities associated with the transport of calcium across several other types of membrane were not affected. The results indicated an essential role for the vacuoles in the regulation of the cytosolic concentration of calcium.
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