- Volume 137, Issue 12, 1991
Volume 137, Issue 12, 1991
- Biochemistry
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Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness
More LessA serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by β-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.
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A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function
More LessEnzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (t d = 1·1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal β-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from β-N-acetylglucosaminidase (β-GlcNAc’ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (K 1 = 175 μm), and non-inhibition by the specific β-GlcNAc’ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6·5–7·0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade preformed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.
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Expression of active yeast pyruvate decarboxylase in Escherichia coli
More LessWe have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter ϕ10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.
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Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity.
More LessA family of peptide antimycotics, termed pseudomycins, has been isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium. These compounds were purified using Amberlite XAD-2 and reverse-phase liquid chromatography. Pseudomycin A, the predominant peptide in a family of four, showed selective phytotoxicity, and had impressive activity against the human pathogen Candida albicans. Amino acid, mass spectroscopic, and comparative electrophoretic and chromatographic analyses revealed that the pseudomycins are different from previously described antimycotics from P. syringae, including syringomycin, syringotoxin and syringostatins. Pseudomycins A–C contain hydroxyaspartic acid, aspartic acid, serine, arginine, lysine and diaminobutyric acid. The molecular masses of pseudomycins A–C, as determined by plasma desorption mass spectrometry, are 1224,1208 and 1252 Da, respectively. Pseudomycin D, on the other hand, has a molecular mass of 2401 Da and is more complex than pseudomycins A–C.
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Effect of chloromethane on veratryl alcohol and lignin peroxidase production by the fungus Phanerochaete chrysosporium
More LessBiosynthesis of veratryl alcohol, a secondary metabolite considered to be an important component of the lignin-degrading system in the fungus Phanerochaete chrysosporium Burds INA-12, was initiated up to 36 h earlier in fungal cultures supplemented with 0·6 and 1·25 mm-CH3Cl compared with unsupplemented mycelia. Peak concentrations of the idiolyte were also about 70% higher in the presence of 0·6 mm-CH3Cl, although peak levels elicited by 0·2, 0·4 and 1·25mm-CH3Cl were lower than in unsupplemented cultures. Advanced initiation of veratryl alcohol biosynthesis in the presence of CH3C1 was reflected in the earlier appearance of lignin peroxidase (LiP) activity. This effect was most noticeable in cultures supplemented with 0·4 and 0·6 mm-CH3Cl, where LiP was evident up to 24 h before detection in unsupplemented cultures. However, peak levels of LiP produced by CH3Cl-augmented mycelia were always less – below 40% in the case of 1·25 mm-CH3Cl – than those recorded in cultures without supplementation. These effects may be explained by restricted metabolic availability of CH3CI as methyl donor for veratryl alcohol biosynthesis in the early stages of fungal growth.
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Origin of oxidized cellulose degradation products and mechanism of their promotion of cellobiohydrolase I biosynthesis in Trichoderma reesei
More LessCellobiono-1,5-lactone (CBL) induces cellulase, particularly cellobiohydrolase I (CBH I) formation in Trichoderma reesei. When used as a sole source of carbon, it promoted comparably poor growth, because (a) the δ-gluconolactone arising by the action of β-glucosidase is not metabolized by the fungus, and (b) it is a low-V max substrate of the T. reesei β-glucosidase. Induction of higher amounts of CBH I were observed when CBL was supplied as carbon source together with cellobiose than when supplied alone. Maximal CBH I levels formed under these conditions were comparable to those when T. reesei was grown on cellobiose plus β-gluconolactone, an inhibitor of β-glucosidase. These data suggest an indirect effect of CBL on CBH I induction, probably by inhibiting cellobiose hydrolysis. The origin of CBL during growth of T. reesei on cellulose was also investigated: aldonic acids and aldonolactones were released from cellulose by T. reesei culture fluids. Artificially oxidized celluloses gave rise to the appearance of higher concentrations of oxidized products but the addition of cellobiose did not, suggesting that their appearance is not due to a cellobiose-oxidizing enzyme. No accumulation of oxidized cello-oligosaccharides occurred when the action of both cellobiohydrolases (CBH I and II) was simultaneously blocked by monoclonal antibodies. These data suggest that cellulose already contains oxidized chain termini, and that these aldonolactones and aldonic acids are released by the action of the two cellobiohydrolases and not by a ‘cellulose-oxidizing’ enzyme.
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- Development And Structure
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Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions
Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (d-GalNAc), galactose (d-Gal), mannose-like residues (d-Man) or l-fucose (l-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the d-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or d-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (d-Gal-specific), although the reactivity of other d-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for d-Man-like residues); however, they were unreactive with the l-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.
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- Ecology
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Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides
More LessOligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5′-end or enzymically at the 3′-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody–enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gramnegative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.
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pH gradients through colonies of Bacillus cereus and the surrounding agar
More LesspH-sensitive microelectrodes, constructed with a tip diameter of about 4 μm, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 μm into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1·45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.
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- Genetics And Molecular Biology
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An ISI-like element is responsible for high-level synthesis of extended-spectrum β-lactamase TEM-6 in Enterobacteriaceae
More LessResistance of Escherichia coli strain HB251 to the newer β-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum β-lactamase TEM-6. The corresponding structural gene, blaT-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that blaT-6 differed by two nucleotide substitutions from blaT-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from blaT-6 resulted in the creation of hybrid promoter P6 in which the –10 region was that of TEM-1 promoter P3 whereas the – 35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.
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Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose
More LessThe rfb region of Yersinia pseudotuberculosis serogroup IIA has been cloned and expression of O antigen in Escherichia coli K12 was demonstrated. Transposon mutagenesis analysis confined the DNA region required for O antigen expression to a 19·3 kb fragment, and the O antigen expressed was visualized by SDS-PAGE and silver staining. Southern hybridization analysis demonstrated significant levels of similarity between the Yersinia rfb region and the 3,6-dideoxyhexose pathway genes rfbF and rfbG, previously isolated from Salmonella enterica LT2, but no similarity to the abequose synthase gene rfbJ of the same strain or the paratose synthase gene rfbS isolated from S. enterica Ty2. The evolutionary relationship between the abequose biosynthetic genes of the two species of Salmonella and Yersinia is discussed.
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Transformation and allelic replacement in Francisella spp.
More LessWe describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.
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Unique DNA plasmid pRS64 associated with chromosomal DNAs of the plant pathogenic fungus Rhizoctonia solani
More LessUnique DNA sequences homologous to the linear DNA plasmid pRS64 were investigated in chromosomal DNAs of isolates belonging to anastomosis group 4 (AG-4) of the plant pathogenic fungus Rhizoctonia solani. Chromosome-sized DNAs of isolates RI-64 and 1271 of AG-4 were separated into six bands by orthogonal-field-alternation gel electrophoresis and hybridized to a cloned segment of pRS64. A small chromosome-sized DNA band of approximately 1·1 Mb carried the sequences homologous to pRS64 DNA. Sequences homologous to pRS64 were also maintained within the chromosomal DNA of isolate 1271 of AG-4 which does not possess the plasmid. The plasmid showed no homology to the mitochondrial DNA of isolate 1271. The possibility that the linear plasmid pRS64 may act as a transposable genetic element is discussed.
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Duplication of both xyl catabolic operons on TOL plasmid pWW15
More LessPseudomonas fluorescens MT15 is the host of the large (250 kbp) TOL plasmid pWW15. We have shown by a combination of hybridization, molecular cloning and enzyme assay that pWW15 carries two distinct regions which share homology with the upper pathway operons (xylCMABN) of other TOL plasmids and two distinct regions which are homologous to the meta pathway operons (xylXYZLTEGFJQKIH) of other TOL plasmids. Both the areas of homology to the upper pathway operons appear to carry all of the structural genes for the three catabolic enzymes of the operon. One of the regions of meta pathway operon homology encodes a complete functional pathway, but the second is incomplete and appears to carry only the genes from xylF downstream.
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- Immunology
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Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro
More LessMultiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK–like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E : T) ratio- and time-dependent. Maximal levels of anti-C albicans activity (approximately 60%) were observed after 4 h and at E : T ≥ 300:1. Similar patterns of anti-C albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA–LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA–LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA–LAK cells (E : T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA–LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.
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- Pathogenicity And Medical Microbiology
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Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene
Lecithin-dependent haemolysin (LDH) of Vibrio parahemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1·5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically crossreactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.
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Influence of capsular neuraminic acid on properties of streptococci of serological group B
More LessNeuraminic acid is thought to be a critical virulence factor of group B streptococci. The present study was designed to further characterize a previously described type III group B streptococcus and its transposon-mutagenized asialo capsular mutant. The wild-type group B streptococcus grew as short chains with a uniform turbidity and had diffuse colonies in soft agar media. In contrast, the asialo mutant grew in fluid media as a granular sediment, formed significantly longer chains and had compact colonies in soft agar. These differences, possibly related to the surface charge of the bacteria, could also be demonstrated in salt aggregation tests and hexadecane adherence studies. The wild-type group B streptococcus showed hydrophilic, and the asialo mutant hydrophobic surface properties. Removal of neuraminic acid from the wild-type strain changed the surface properties from hydrophilic to hydrophobic. A similar masking effect of capsular neuraminic acid could be observed in adherence and phagocytosis experiments. In contrast to the wild-type strain, the asialo mutant adhered significantly more to buccal epithelial cells and was phagocytosed more by polymorphonuclear leucocytes. These altered properties might possibly be of importance for group B streptococcal pathogenicity.
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Distinctions in DNA and protein profiles among clinical isolates of Mycoplasma pneumoniae
More LessClinical isolates of Mycoplasma pneumoniae previously shown to exhibit significant sequence divergency in a major 170 kDa adhesin, designated P1, were further characterized using restriction enzyme fingerprinting of genomic DNA and two-dimensional gel electrophoresis of total proteins. Numerous differences in DNA restriction patterns and protein profiles were found, possibly reflecting various degrees of virulence and antigenic potential.
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Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1
More LessEach of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidinagarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (~ 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.
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Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions
More LessMonoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.
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- Physiology And Growth
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Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate
More LessThe chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10–20 min, whereas the wild-types took 45–60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (M r < 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.
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Phenotypic variation of Pseudomonas putida and P. tolaasii affects attachment to Agaricus bisporus mycelium
More LessThe effect of phenotypic variation on attachment of Pseudomonas tolaasii and P. putida to Agaricus bisporus mycelium was investigated. Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A. bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed. This was most pronounced in P. tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant. The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach. Attachment of the wild-type form of P. putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces. No correlation was observed between bacterial hydrophobicity and ability to attach to A. bisporus mycelium. Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium. Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface. A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells. This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.
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Behaviour of Azospirillum brasilense in a spatial gradient of oxygen and in a ‘redox’ gradient of an artificial electron acceptor
More LessAzospirillum brasilense responds to a spatial gradient of oxygen by formation of a band in the zone of optimal oxygen concentration. The same behaviour was observed in a ‘redox’ gradient of tetramethyl-p-phenylenediamine (TMPD) oxidized by ferricyanide, under conditions where oxygen uptake was inhibited by cyanide. Inhibitors of the bc 1-complex of the redox chain did not inhibit aerotaxis. Cells inhibited by a high cyanide concentration still showed slow motility and weak attraction to oxygen, possibly as a result of cyanide-resistant respiration (3–5% of the initial level). It is suggested that electron flow through the redox chain (and/or consequent changes in protonmotive force), rather than binding of oxygen to terminal oxidases, may be a signal in the aerotactic response of A. brasilense.
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Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR
More LessTo investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5·6 mgl–1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP 3.BR appeared to be the only one that arrested at the end of the exponential phase of growth. At least 15 proteinases (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF-39·5, PF-38·5, PF-36·5, PF-25·5 and PF-20·5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.
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Escherichia coli metabolism in space
More LessSUMMARY: Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment – weightlessness and heavy particle radiation – on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.
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Effects of alcohols on the respiration and fermentation of aerated suspensions of baker’s yeast
More LessThe immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker’s yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0–20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.
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Examination of the role of Na+ in the physiology of the Na+-dependent soil bacterium Azotobacter salinestris
More LessThe growth of the Na+-dependent soil bacterium Azotobacter salinestris strain 184 was inhibited only 36% by 50 μm-carbonyl cyanide m-chlorophenylhydrazone (CCCP) at alkaline pH, whereas other species of this genus were inhibited 80–90% under the same conditions. Growth of strain 184 at alkaline pH was inhibited 66% by 50 μm-monensin and 100% by monensin plus CCCP. The majority of the ATPase activity on everted membrane vesicles prepared from strain 184 grown at alkaline pH was sensitive to azide and N,N′-dicyclohexylcarbodiimide (DCCD), but ATPase was less sensitive to these inhibitors when Na+ was present. The respiratory activity of strain 184 was neither dependent on nor activated by Na+ and was unaffected by the antagonistic Na+-analogues K+ and Rb+. A Na -dependent, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) supersensitive NADH oxidase was not present in strain 184. Na+ was required in the growth medium to promote optimal cell yields. Limiting the amount of Na+ available caused a lag phase in which cell viability was lost. Viability was maintained by the addition of Li+ or Mg2+ to Na+-limited medium, but only Li+ appeared to promote growth. K+ appeared to be a competitive inhibitor of a Na+/Li+ site required for cell growth. Rb+ was a more complex competitor and affected the final yield and the growth rate of strain 184. Rb+-tolerant mutants of strain 184 were selected and the majority of these were found to be defective in the Na+-dependent acid excretion normally observed with A. salinestris. Analysis of an acid over-producing strain showed that Rb+ appeared to be an uncompetitive inhibitor of Na+-dependent growth and in competition with Na+ as a promoter of acidification.
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Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways
C. Tricot, V. STALON and C. LEGRAINPseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.
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Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules
More LessBacteroids were isolated anaerobically from root nodules of soybean (Glycine max L. Merr.) and supplied with 14C- or 15N-labelled glutamate or aspartate under microaerobic conditions where bacteroids retained a high activity of N2 fixation. Glutamate and aspartate significantly stimulated N2 fixation (C2H2 reduction) and respiration (CO2 evolution) of isolated bacteroids. They were both utilized as substrates for bacteroid respiration and their nitrogen was rapidly released from the bacteroids as NH3. Amino-oxyacetate (AOA) strongly inhibited glutamate utilization, but aspartate utilization was only slightly affected by AOA. Substantial activity of aspartate ammonia-lyase (aspartase) was detected in the cell-free extract of bacteroids. These results suggest that the major pathway of glutamate utilization in bacteroids is transamination to form aspartate followed by direct deamination of aspartate by aspartase. It is also suggested that the 4-aminobutyrate pathway (GABA-shunt) is partly responsible for the catabolism of glutamate by soybean nodule bacteroids. The results are discussed in terms of the possible relationships between C4-dicarboxylates and glutamate in carbon and nitrogen metabolism in soybean nodule bacteroids.
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- Systematics
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Epidemiological analysis of a methicillin-resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA
More LessThe genomic DNA of 58 isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained during an infection outbreak at two major Canberra hospitals was analysed for restriction fragment length polymorphism (RFLP) by digestion with the endonuclease SmaI and resolution of the fragments by pulsed-field gel electrophoresis. Based on the fraction of common fragments generated by the endonuclease, DNA similarities among the isolates were estimated. Distance matrix analysis showed that the MRSA isolates could be divided into two major clusters (RFLP types I and II) and one minor one (type 46). A fourth group of miscellaneous isolates was found to be heterogeneous in terms of DNA sequence similarity. The epidemiological data indicated that RFLP type I was most common in the intensive care units in the two hospitals, with particular subtypes of RFLP type I concentrated in individual units. RFLP type II and the miscellaneous group were more generally distributed. Type 46 isolates appear to be related to a group which was present in epidemics in Melbourne hospitals in the early 1980s. Using the standard phage set, the RFLP type I group was largely untypable. However, type II isolates were all phage typable, with a shared susceptibility to phages 29/85/95/90; type 46 isolates had a shared susceptibility to phages 85/90. The miscellaneous isolates were of variable phage types.
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