- Volume 139, Issue 9, 1993
Volume 139, Issue 9, 1993
- Review Article
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Microbial cleavage of nitrate esters: defusing the environment
More LessSUMMARY: The products of condensing organic hydroxyl groups (ROH) with the mineral acids (hydrochloric, nitric, phosphoric and sulphuric) collectively constitute a major part of the output of the synthetic organic chemical industry, with a wide diversity of applications including surfactants, pesticides, herbicides, dyes, methylating agents, explosives and pharmaceuticals. Compounds containing similar or related structures also occur as natural products. Phosphate esters are, of course, exceptional for their ubiquity in the living world, ranging from the simple intermediary metabolites of glycolysis, through phospholipids, to the backbone of DNA. Sulphate esters too are abundant (Dodgson et al., 1982) and naturally occurring halogenated compounds are also being detected in increasing numbers (for examples, see Strunz, 1984; Engvild, 1986; Neidleman & Geigert, 1986; Harper & Hamilton, 1988). It is therefore no surprise that living organisms have evolved phosphatase (Boyer, 1971), sulphatase (Dodgson & Rose, 1975; Dodgson et al., 1982) and dehalogenase (Neilson, 1990; Hardman, 1991) enzyme systems for initiating biodegradation of such compounds by the removal of the mineral moiety. In marked contrast, we are unable to find any examples of naturally occurring nitrate esters, so that the introduction of such compounds into the environment during their industrial production and usage constitutes a true xenobiotic challenge to microorganisms. This raises intriguing questions about microbial capability for biotransformation/biodegradation of nitrate esters, not only from an academic viewpoint but also because of the wide industrial usage of these compounds and their likely impact on the environment.
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- Biochemistry
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A novel pathway for the catabolism of 4-nitrotoluene by Pseudomonas
More LessSUMMARY: Eleven strains of Pseudomonas were isolated by selective enrichment on 4-nitrotoluene (4NT). They all utilized 4NT, 4-nitrobenzyl alcohol (4NBA) or 4-nitrobenzoate (4NBZate) as sole sources of carbon and nitrogen. One strain, TW3, was used for more detailed studies. 4NT-grown cells of TW3 take up O2 when incubated in the presence of 4NBA, 4-nitrobenzaldehyde (4NBZ) and 4NBZate. HPLC analysis of culture supernatants showed that 4NBZ and 4NBZate were formed when 4NT-grown cells were incubated with 4NBA, whereas only 4NBZate was found when they were incubated with 4NBZ. Two dehydrogenases were detected in extracts of 4NT-grown cells. 4NBA dehydrogenase could be assayed by a dye-linked assay whereas 4NBZ dehydrogenase activity was linked to NAD+ reduction. No nitrite was detected in supernatants of 4NBZate-grown cells incubated with 4NBZate but the nitrogen appeared as ammonium. The only aromatic ring-cleavage dioxygenase that was induced during growth on the nitroaromatics was protocatechuate 3,4-dioxygenase. It is proposed that the pathway for 4NT catabolism proceeds via 4NBA, 4NBZ and 4NBZate and ultimately to protocatechuate with release of the nitro group as ammonium.
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Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides
More LessSUMMARY: A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed a and , which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the a and peptides. From the N-terminal end, 21 and 22 amino acid residues of a and , respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, a and may be encoded by the same gene, since a appeared to be a truncated form of . Alanine, the first amino acid residue at the N-terminal end of was not present at this position in a. Otherwise the sequences of a and appeared to be identical. The calculated molecular masses of the sequenced part of a and were 2426 and 2497 Da, respectively. The molecular masses of a and as determined by mass spectroscopy were 2687 30 and 2758 30 Da, respectively, indicating that (i) the only difference between a and was the presence of the N-terminal alanine residue in , and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the a and peptides. Both a and may form amphiphilic a-helices, suggesting that they are pore-forming peptides that create cell membrane channels through a barrel-stave' mechanism.
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Formaldehyde dismutase activities in Gram-positive bacteria oxidizing methanol
SUMMARY: Extracts of methanol-grown cells of Amycolatopsis methanolica and Mycobacterium gastri oxidized methanol and ethanol with concomitant reduction of N, N'-dimethyl-4-nitrosoaniline (NDMA). Anion-exchange chromatography revealed the presence of a single enzyme able to catalyse this activity in methanol- or ethanol-grown cells of M. gastri. A. methanolica, however, possessed two different enzymes, one of which was similar to the single enzyme found in M. gastri. The methanol: NDMA oxidoreductases (MNO) were purified to homogeneity from methanol-grown cells of A. methanolica and M. gastri. Both enzyme preparations showed similar relative molecular masses with subunits of M r 50000 and 49000, and native enzymes of M r 268000 and 255000 (gel-filtration data for A. methanolica and M. gastri, respectively). Both enzymes also displayed a similar substrate specificity. They were active with methanol and various other primary alcohols (yielding the corresponding aldehydes), polyols and formaldehyde. In addition, the MNO enzymes produced methylformate from methanol plus formaldehyde, and catalyzed formaldehyde dismutase and NADH-dependent formaldehyde reductase reactions. They did not possess NAD(P)+- or dye-linked alcohol dehydrogenase or oxidase activities.
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Xylanases from Streptomyces cyaneus: their production, purification and characterization
More LessSUMMARY: Streptomyces cyaneus produces extracellular xylanases in good yield when grown on ball-milled straw and xylan. Ball-milled straw induced nearly twice as much of these activities as did xylan, but phenolic substances released from the straw impeded purification of enzymes. Three extracellular xylanases, I, II and III, were identified in xylan-grown culture supernatants. Xylanases I and II were each purified to give single bands by SDS-PAGE. They had M r values of 37500 and 34000 respectively, while that of III was apparently 45000. pI values were 5.1 (I) and 5.3 (II) while pH and temperature optima, in 10 min assays, were 8.5, 72° (I) and 6.5, 65 °C (II).
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Purification and partial characterization of thiosulphate dehydrogenase from Thiobacillus acidophilus
SUMMARY: Thiosulphate dehydrogenase (EC 1.8.2.2; thiosulphate:acceptor oxidoreductase) was purified to apparent homogeneity from Thiobacillus acidophilus by a combination of ammonium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration. The enzyme catalysed the oxidation of thiosulphate (S2O2- 3) to tetrathionate (S4O2- 6) with potassium ferricyanide as an artificial electron acceptor. The molecular mass of the native enzyme, as determined by gel filtration, was 102 ± 4.2 kDa. The enzyme contained two different subunits with a molecular mass of 24 ± 0.9 and 20 ± 1.0 kDa (SDS-PAGE), respectively. Both subunits contained c 553-type haem with absorption bands at 553, 524 and 416 nm. A 77 K spectrum of purified thiosulphate dehydrogenase revealed that the absorption at 553 nm is due to different haem groups. A cytochrome content of 5.3 mole c-type haem per mole of native enzyme was calculated. The pH optimum of the purified enzyme was 3. Apart from ferricyanide, Wurster's blue (the free radical of tetramethyl p-phenylenediamine) and horse heart cytochrome c could also serve as electron acceptors, though less effectively than ferricyanide. At pH 7.0, the K m for thiosulphate was 0.54 mM. The K m could not be determined at the pH optimum due to the chemical reactivity of thiosulphate at low pH values. Sulphite was a potent inhibitor of enzyme activity.
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Synthesis in vitro of crystalline chitin by a solubilized enzyme from the cellulosic fungus Saprolegnia monoica
More LessSUMMARY: Enriched preparations of chitin synthase were obtained from cell homogenates from Saprolegnia monoica. Chitin synthase was solubilized from a mixed membrane fraction by two successive digitonin treatments. Glycerol gradient centrifugation of the solubilized proteins separated the chitin synthase activity from the majority of proteins and from -1,3 and -1,4 glucan synthases. The properties of chitin synthase from this Oomycete fungus are similar to those reported for the enzymes of chitinous fungi. The solubilized enzyme catalysed the synthesis in vitro of spindle-like crystals of chitin. Biophysical analysis (by electron and X-ray diffractometry and infrared spectroscopy) demonstrated that the polymer synthesized in vitro was a-chitin. These results and those previously reported demonstrate unambiguously that chitin synthase and chitin are normal components of the cell wall of the cellulosic fungus S. monoica.
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The cell wall of the oleaginous yeast Trichosporon cutaneum
More LessSUMMARY: The cell wall of Trichosporon cutaneum consists of 11% protein, 63% neutral carbohydrate, 9% glucosamine and 13% glucuronic acid. The sugars include glucose (32%), mannose (6%) and traces of xylose and galactose. The cell wall was fractionated with alkali to yield a mixture of alkali-soluble matrix components, and an alkali-insoluble glucan associated with chitin. The alkali-insoluble glucan contained a mixture of (1-3) and (1-6) glycosidic linkages. It was only partly susceptible to digestion by the β(1-3) glucanase, Zymolyase. The alkali-soluble fraction contained glucan, mannan and acidic polymers. The glucan was (1-3)-linked with no (1-6) linkages and only trace amounts of (1-3-6)-linked glucose. It was resistant to digestion by Zymolyase. Extensive hydrolysis of this fraction with trifluoroacetic acid released a high-molecular-mass glucuronan which had 1H- and 13C-NMR profiles matching those of the β(1-4) glucuronan, mucoric acid. Xylomannan was purified from isolated cell walls and from whole cells. It contained glucose, mannose, xylose, and D-glucuronic acid. It was very similar in composition and structure to the capsular polysaccharides of Cryptococcus neoformans, and to an extracellular polysaccharide produced by another yeast described as T. cutaneum. Electron microscopy showed that the cell wall of T. cutaneum has a lamellar structure characteristic of a basidiomycetous yeast rather than the electron-dense ‘fuzzy coat’ seen in Candida albicans.
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Purification and characterization of glutathione reductase from Chlamydomonas reinhardtii
More LessSUMMARY: Glutathione reductase was purified to homogeneity from the unicellular alga Chlamydomonas reinhardtii. The enzyme was a monomer with a molecular mass of 54-56 kDa as judged by gel filtration and SDS-PAGE. The activity was maximal at pH 8.2 and 49 °C. The enzyme was specific for NADPH, but not for NADH. The reverse reaction with NAD(P)+ and GSH (glutathione, reduced form) was not observed in the pH range 4.8-8.2. K m values for NADPH and GSSG (glutathione, oxidized form) were 10.6 μ;m and 54.1 μ;M, respectively. Thiol inhibitors and metal ions such as Hg2+ and Cu2+ markedly inhibited the enzyme activity. Activity was lost when the apoenzyme was prepared by dialysis, but was restored to 40% of the original activity by the addition of 50 μ;M-FAD. The enzyme reaction proceeded via a branching mechanism. Upon immunoprecipitation, glutathione reductase activity of C. reinhardtii was inhibited 50% and 90% by antibodies generated against spinach and Euglena glutathione reductases, respectively. Both antibodies cross-reacted with C. reinhardtii glutathione reductase in an immunoblot analysis.
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- Biotechnology
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Salt-stimulation of caesium accumulation in the euryhaline green microalga Chlorella salina: potential relevance to the development of a biological Cs-removal process
More LessSUMMARY: Accumulation of Cs+ by Chlorella salina was 28-fold greater in cells incubated in the presence than in the absence of 0.5 M-NaCl. An approximate 70% removal of external Cs+ resulted after 15 h incubation of cells with 50 μ;M-CsCl and 0.5 M-NaCl. LiCl also had a stimulatory effect on Cs+ uptake, although mannitol did not. Cs+ influx increased with increasing external NaCl concentration and was maximal between 25-500 mM-NaCl at approximately 4 nmol Cs+ h−1 (106 cells)−1. Little effect on Cs+ uptake resulted from the presence of Mg2+ or Ca2+ or from varying the external pH, and Cs+ was relatively non-toxic towards C. salina. At increasing cell densities (from 4 × 105 to 1 × 107 cells ml+1), decreasing amounts of Cs+ were accumulated per cell although the rate of Cs+ removal from the external medium was still greatest at the higher cell densities examined. Freely suspended C. salina and cell-loaded alginate microbeads accumulated similar levels of Cs+, however, 46% of total Cs+ uptake was attributable to the calcium-alginate matrix in the latter case. When Cs+-loaded cells were subjected to hypoosmotic shock, loss of cellular Cs+ occurred allowing easy Cs+ recovery. This loss exceeded 90% of cellular Cs+ when cells were washed with solutions containing ≤ 50 mM-NaCl between consecutive Cs+ uptake periods; these cells subsequently lost their ability to accumulate large amounts of Cs+. Maximal Cs+ uptake (approximately 85.1% removal after three 15 h incubations) occurred when cells were washed with a solution containing 500 mM-NaCl and 200 mM-KCl between incubations. The relevance of these results to the possible use of C. salina in a salt-dependent biological Cs-removal process is discussed.
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- Development And Structure
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Ultrastructure of proteinase-secreting cells of Candida albicans studied by alkaline bismuth staining and immunocytochemistry
More LessSUMMARY: The ultrastructure of Candida albicans cells induced to secrete extracellular proteinase (EPR) has been studied. Electron microscopy employing alkaline bismuth staining, a method which stains polysaccharides, clearly revealed Golgi-like bodies and secretory vesicles in C. albicans cells. After EPR induction, there was no apparent increase in the number of these structures. Instead, many flocculent granules appeared at the periphery of induced cells. The granules were similar to secretory vesicles in size, but were more irregular in shape. Similar granules were observed in non-induced cells, though less frequently than in induced cells. Brefeldin A, a specific inhibitor of membrane transport in the secretory pathway, caused the accumulation of EPR and Golgi-like bodies in EPR-induced cells, but did not affect the accumulation of the granules. These results suggest that the granules are unrelated to EPR secretion. Electron microscope immunocytochemistry with affinity-purified anti-EPR antibodies showed that the granules in EPR-induced cells were recognized by the antibodies. This recognition was completely inhibited by the presence of glycogen, suggesting that antibodies cross-react with glycogen-like polysaccharides in the granules. Although the location of EPR within the cells remains unclear, the results suggest that EPR might be secreted via the constitutive secretory pathway, and that EPR is glycosylated to give a structure with some similarity to glycogen.
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- Environmental Microbiology
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Small-scale patchiness in the chemistry and microbiology of sediments in Lake Geneva, Switzerland
More LessSUMMARY: Lake Geneva is a large, holomictic, eutrophic lake with a maximum depth of about 300 m. The sediments in the central basin have a pillow-like appearance. The soft elevations containing the major portion of the recently sedimented detritus are separated by trenches of 5 to 15 cm depth in which the top sediment layers seem to be missing. Bottom-dwelling fishes (Lota lota) prefer the trenches as their habitat and might partly be responsible for the turbation of the trench sediment layers. Thus, within distances of 10 to 30 cm two sediment types can clearly be distinguished. They differ with respect to morphology and chemical stratification. Concentration depth profiles of nitrate, manganese(II), iron(II), sulphate, and methane dissolved in the interstitial water reveal the location within the sediment of microbially catalysed redox processes. The redox transition zone (RTZ) from aerobic to anaerobic is located only a few millimetres below the sediment surface in the pillow sediments, which contain the bulk of the organic detritus. The RTZ is at a depth of approximately 6 cm in the trench sediments, which are poor in oxidizable organic matter. The same thermodynamic sequence of microbially catalysed redox reactions can be observed in both sediment types. As a consequence, microbial activities as well as diffusion fluxes of dissolved substances in and out of the different sediment regions vary greatly. This leads to small-scale horizontal differences in the sediment's abilities to supply nutrients to the bottom water, which is probably a major controlling factor for sediment-borne eutrophication of this lake.
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Antibody and DNA probes for detection of nitrite reductase in seawater
More LessSUMMARY: A polyclonal antiserum was produced by immunization with nitrite reductase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was nearly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sediment environments were also screened; several isolates from an intertidal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently very similar NiR proteins did not react with the antiserum. These results imply that the NiR protein is more variable even among closely related strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. stutzeri (ATCC 14405) DNA and used to screen denitrifying strains and isolates. The probe hybridized with a greater variety of strains than did the antiserum, implying that the DNA probe may be a more broadly useful and functional probe in environmental samples, whilst the NiR antiserum is nearly strain- or, at most, species-specific. Limits for detection of the enzyme and gene in seawater were estimated and NiR DNA was detected in DNA extracted from natural seawater. The hybridization data imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.
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- Genetics And Molecular Biology
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Transposition of the TOL catabolic genes (Tn4651) into the degradative plasmid pSAH of Alcaligenes sp. O-1 ensures simultaneous mineralization of sulpho- and methyl-substituted aromatics
More LessSUMMARY: Mixtures of 2-aminobenzenesulphonate (trivial name orthanilic acid, OA) and 3-methylbenzoic acid (3-MB), which are degraded by enzymes of plasmid-encoded pathways, can exert inhibition of growth and respiration in Alcaligenes sp. O-1 and Pseudomonas putida mt-2 depending on the ratio of their concentrations. The pronounced inhibition of Alcaligenes sp. O-1 growing on OA by the addition of equimolar amounts of 3-MB is characterized by a rapid inactivation of the OA-converting desulphonation activity. The exconjugant Alcaligenes sp. O/T was selected for simultaneous breakdown of OA and 3-MB by assembling the catabolic pathways from the plasmids pSAH (OA) and pWW0 (3-MB) of the above strains. The transpositional insertion of the TOL catabolic genes (Tn4651) from pWW0 into the recombinant plasmid of the exconjugant O/T was detected by Southern blot hybridization using the TOL plasmid as a probe. The exconjugant showed a rapid inactivation of OA desulphonation activity similar to the parent strain. However, following induction of the TOL catabolic genes and mineralization of 3-MB, the exconjugant O/T recovered and displayed high desulphonation activity, thus allowing sequential breakdown of both substrates. Our results clearly extend the expression range of the TOL catabolic genes, but not the replication ability of the plasmid, to the genus Alcaligenes.
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Sequence and expression of the Mycobacterium leprae dnaJ gene
More LessSUMMARY: Study of Mycobacterium leprae, the causative agent of leprosy, has been advanced by the isolation of genes encoding mycobacterial proteins including dnaK encoding the M. leprae 70 kDa heat shock protein. The sequence downstream from dnaK revealed a second open reading frame coding for a protein of 389 amino acids with a calculated molecular mass of 41.2 kDa. Sequence analysis demonstrated significant DNA homology with the dnaJ gene of other organisms. High amino acid sequence identity was obtained between the DnaJ protein of M. leprae and M. tuberculosis (89 %) with significant divergence between the two occurring only at the C-terminal end. The expressed recombinant DnaJ protein had a molecular mass of 42 kDa.
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Catabolite repression of β-glucanase synthesis in Bacillus subtilis
More LessSUMMARY: β-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate:sugar phosphotransferase system is not involved in carbon catabolite repression of β-glucanase synthesis. Carbon catabolite repression of β-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of β-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both β-glucanase and α-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.
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The giant linear plasmid pHG207 from Rhodococcus sp. encoding hydrogen autotrophy: characterization of the plasmid and its termini
More LessSUMMARY: As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids - on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.
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Conserved and variable regions in protein Arp, the IgA receptor of Streptococcus pyogenes
More LessSUMMARY: The streptococcal M protein family, a number of cell surface molecules that interact with the human immune system, can be divided into two major classes, A and C, characterized by different types of repeats in the central part of the molecule. Class A and class C molecules are known to have a variable N-terminal region and a more conserved C-terminal region, but little is known about the mechanisms that give rise to this structural variation. In this report, we show that two variants of protein Arp, an IgA receptor in class C of the M protein family, have virtually identical signal sequences and C-terminal halves, but unrelated N-terminal sequences. Comparison of the sequences of the two genes and their flanking regions also demonstrates the presence of well-defined variable and conserved regions. Our results strongly suggest that the N-terminal sequence variation between the two variants of protein Arp was generated through an intergenic recombination event, rather than through intragenic recombination or accumulation of mutations.
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Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae
More LessSUMMARY: Metarhizium anisopliae isolates from several insect hosts and from various sugar cane growing areas of Queensland, Australia, were examined for genetic diversity using random amplified polymorphic DNA (RAPD) markers. Thirty isolates of M. anisopliae var. anisopliae and one isolate of M. anisopliae var. majus were examined. Ten randomly chosen 10mer or 11mer primers were used and RAPD banding patterns were compared. Thirty distinct genotypes could be distinguished amongst the 31 isolates tested on the basis of RAPD patterns. Six of the isolates classified as M. anisopliae var. anisopliae exhibited closer similarity to the M. anisopliae var. majus isolate than to other anisopliae strains tested. Isolates exhibiting similar (> 80 % similarity) RAPD profiles tended to be isolated from the same geographic area and evidence for the persistence of particular fungal genotypes in specific geographical localities was obtained. Pathogenicity assays suggested that, in some instances, RAPD groupings may also indicate insect host range. The mean similarity amongst isolates measured by band sharing in all pairwise comparisons was 41% and the most distinct pair of isolates shared only 9% of their RAPD bands. We conclude that the isolates tested belonging to the species M. anisopliae, as assessed on morphological grounds, represent a very diverse genetic group. The results also suggest that RAPD markers may be useful for the tracking of specific biocontrol strains in the field.
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The Sc7/Sc14 gene family of Schizophyllum commune codes for extracellular proteins specifically expressed during fruit-body formation
SUMMARY: The Sc7 and Sc14 genes are specifically expressed in the dikaryon of the basidiomycete fungus Schizophyllum commune during fruiting. These genes are closely linked (within 6 kb) and highly similar in gene structure and nucleotide sequence (70% identical nucleotides in their coding regions). The encoded proteins (204 and 214 amino acids, respectively) have 87% similarity in amino acids (56% of the amino acids are identical). They contain putative signal sequences for secretion, are rich in aromatic amino acids which are generally located at similar positions, and they are generally hydrophilic. Inspection of databanks showed similarities with pathogenesis-related proteins (PR1) from plants, testis-specific proteins from mammals and venom allergen proteins from insects. An antibody raised against a Sc7 fusion protein showed the presence of the Sc7 protein in the culture medium and in the fruit bodies where it is apparently loosely associated with hyphal walls.
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Molecular analysis of a gene encoding a serum-resistance-associated 76 kDa surface antigen of Haemophilus somnus
More LessSUMMARY: Haemophilus somnus is a Gram-negative bacterial bovine pathogen which can cause disease or be carried asymptomatically. We previously showed that four serum-sensitive isolates from asymptomatic carriers lacked a 13.4 kb sequence of chromosomal DNA that was present in two virulent serum-resistant strains. We have since sequenced 5 kb of the 13.4 kb fragment from a serum-resistant strain, which contained an open reading frame (ORF) of at least 4.5 kb. From Western blot analysis, the ORF was shown to encode a 76 kDa protein (p76) that co-migrated with a 76 kDa H. somnus surface protein. Both the recombinant and natural p76 reacted with convalescent-phase serum from a cow in an experimental H. somnus abortion study. The translational start site for p76 was identified by deletion analysis of subclones of the 5 kb cloned sequence. The 4.5 kb ORF contained 1.2 kb tandem direct repeats (DRs), with 65% identity between the two repeats at the protein level. The 5' DR (DR1) included the start site for the 76 kDa protein, and DR2 had a flanking inverted repeat, suggestive of an insertion-sequence-like element.
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Comparison of PCR fingerprinting, by random amplification of polymorphic DNA, with other molecular typing methods for Candida albicans
More LessSUMMARY: Fingerprinting by random amplification of polymorphic DNA (RAPD) was compared with existing molecular typing systems for Candida albicans. Fifteen isolates were chosen, including three from the same patient; these gave 14 distinct karyotypes by pulsed-field gel electrophoresis (PFGE) and 7 different DNA types by EcoRI-generated restriction fragment length polymorphisms (RFLPs). RAPD with primer I (5' GCT GGT GG3') gave 5 types, whereas primer II (5' GCG CAC GG3') yielded 11 types. Combining the results from both primers, all isolates were unique by RAPD with the exception of the three from the same patient. RAPD provided a fast, economical and reproducible means of typing C. albicans with a level of discrimination approaching that of PFGE.
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- Pathogenicity And Medical Microbiology
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Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation
More LessSUMMARY: Porphyromonas gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6.9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 °C. A 46 kDa OMP was observed in haeminexcess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 °C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells. The major HBP (mol. mass 32.4 kDa) corresponded to a similar sized Kenacid-blue-stained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.
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Chlamydia trachomatis infection of cultured motile cells after uptake of chlamydiae from the substratum
More LessSUMMARY: The ability of motile cells to remove small inanimate particles from solid substrata is well documented. We show here that motile cells will pick up and internalize infectious particles of the obligate intracellular parasite Chlamydia trachomatis when they are adherent to the substratum over which the host cells move. Two cell types were used to assess chlamydial uptake; a feeder independent human squamous cell carcinoma variant (AC3A cells) and the McCoy cell line. Purified chlamydial elementary bodies were attached to glass or collagen-coated glass by centrifugation. Suspensions of cells were then allowed to sediment on to the substrata to which chlamydiae had attached. Both types of cell picked up chlamydiae and transported them over their surface during the course of attachment and spreading. Stereoscopic images obtained by confocal microscopy demonstrated that chlamydiae were found mainly on the surface of non-spread cells. After the cells had spread on the substratum they began to move around forming tracks where the chlamydiae had been removed. Some cell-surface-attached chlamydiae were endocytosed and a proportion of these proliferated during the 48 h after plating. However, chlamydiae attached to the substratum lost infectivity by a simple exponential decay process within a few hours of incubation in the extracellular environment. Therefore, increasing numbers of non-viable organisms were probably endocytosed as the time of extracellular incubation increased. This mode of infection may be relevant to in vivo situations where cell migration occurs after damage to mucosal surfaces.
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Purification of yersiniabactin: a siderophore and possible virulence factor of Yersinia enterocolitica
More LessSUMMARY: HPLC analysis revealed that Yersinia enterocolitica WA-C produced two substances under iron-limiting conditions one of which was identified as 2,3-dihydroxybenzoyl-L-serine. The other compound had iron-complexing activity and was called yersiniabactin. The fur mutant H1852 was shown to produce yersiniabactin constitutively in an iron-independent manner. Yersiniabactin was isolated by ethyl acetate extraction from the spent medium of H1852, size-fractionation chromatography and preparative HPLC. A catechol function was demonstrated with different chemical assays and by UV-visible spectroscopy. The molecular mass of yersiniabactin was determined to be 482 Da. Purified yersiniabactin stimulated growth of Y. enterocolitica and Escherichia coli Ø under iron-limiting conditions and apparently served as an iron carrier. Transport of 55Fe-yersiniabactin was TonB-dependent, indicating a receptor-mediated uptake across the outer membrane. A pesticin-resistant mutant missing the receptor protein FyuA was unable to transport and use yersiniabactin as a siderophore.
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Biological activities of cell envelope fragments of the archaeobacterium Sulfolobus solfataricus: lethal toxicity, local hypersensitivity, pyrogenicity and spleen lymphocyte mitogenicity
SUMMARY: The sensitizing effect and the local and general toxicity related to membrane components of the archaeobacterium Sulfolobus solfataricus was studied. Cell envelope fragments were biologically active but this activity was lost upon separation of the lipid and protein components. The envelope fragments exerted lethal effects on mice sensitized with D-galactosamine that were prevented by pretreatment with anti-TNF-α serum. This lethal activity occurred in both LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mouse strains. In addition, Sulfolobus envelope fragments tested in rabbits caused a local Shwartzman reaction, and showed pyrogenic activity. In vitro, the envelope fragments that act on spleen lymphocytes of the LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mice caused an uptake of [3H]thymidine similar to that caused by concanavalin A. A similar toxic activity to that exerted by eubacteria is therefore exerted by this non-pathogenic archaeobacterium despite the difference in surface chemistry.
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A haemagglutinating adhesin of group B streptococci isolated from cases of bovine mastitis mediates adherence to HeLa cells
More LessSUMMARY: Rabbit erythrocytes were agglutinated by 43.4 % of group B streptococci isolated from bovines but by none isolated from humans. Haemagglutination was enhanced by cultivation of the bacteria under microaerophilic conditions. Most of the haemagglutinating strains had protein type antigen X, either alone, or in combination with polysaccharide antigens. Heat and proteolytic treatment of the bacteria destroyed the haemagglutination activity. The haemagglutinin could be solubilized from the bacterial surface by mutanolysin treatment and isolated from culture supernatant fluid by ammonium sulphate precipitation. The isolated haemagglutinin did not cause direct agglutination of erythrocytes. However, binding of the haemagglutinin to rabbit erythrocytes could be visualized by agglutination of haemagglutinin-treated erythrocytes by specific antiserum obtained by absorption. Western blotting showed that the haemagglutinin obtained from erythrocyte lysates contained an antibody-reactive band with a molecular mass of 43 kDa. Haemagglutination-positive strains adhered to HeLa cells in higher numbers than did haemagglutination-negative strains. The HeLa cell adherence of Group B streptococci was inhibited in the presence of isolated haemagglutinin or of specific antiserum against the haemagglutinin. These observations suggest that the haemagglutinating adhesins of bovine group B streptococcal isolates are directly involved in the adherence mechanisms of these organisms.
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- Physiology And Growth
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Iron-regulated salicylate synthesis by Pseudomonas spp.
More LessSUMMARY: Two iron-regulated compounds have been found in acidified ethyl acetate extracts from culture supernatants of Pseudomonas aeruginosa and Pseudomonas cepacia type-strains. Synthesis of both compounds paralleled iron-deficient growth, and was repressed in the presence of 100 ;M-FeCl3. Yields of these substances varied among different strains and attained maximum levels during stationary phase. Thin layer chromatographic analysis in five different solvent systems revealed that the slower-moving compound chromatographed as two distinct bands, and showed RF values and spectral properties similar to pyochelin. The faster-moving compound co-migrated as a single band with a standard of commercial salicylic acid in each of the chromatographic systems tested. Moreover, a molecule with an identical RF was also produced by Pseudomonas fluorescens CHA401, which is known to synthesize salicylic acid as the only siderophore during iron-limited growth. Spectrophotometric and spectrofluorometric titrations led to the identification of this iron-regulated compound as salicylic acid, in agreement with the structure deduced from 1H-NMR and mass spectroscopy. The identity of the P. cepacia siderophore azurechelin as salicylic acid was also conclusively demonstrated. Salicylic acid, like pyochelin and pyoverdin, promoted P. aeruginosa growth in an iron-depleted medium. These results are consistent with a putative siderophore activity for salicylic acid, i.e. azurechelin, as has been demonstrated for P. aeruginosa, P. fluorescens and P. cepacia. Thus, salicylic acid is likely to act as a siderophore in more than one species belonging to the genus Pseudomonas.
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Physiological and genetic regulation of rRNA synthesis in Lactococcus
More LessSUMMARY: The macromolecular composition of Lactococcus was regulated by growth rate in the same general way as that of less fastidious bacteria such as Escherichia coli and Salmonella typhimurium. The ratios of RNA:DNA and RNA:protein increased approximately threefold over a 13.5-fold increase in growth rate, whereas the ratio of DNA:protein remained approximately constant. Using reporter genes fused to a DNA fragment of a cloned lactococcal rRNA operon, promoter activity was located upstream of the 16S rRNA structural gene. This DNA fragment had some characteristics typical of a rrn promoter in E. coli. Two consensus promoter sequences P1 and P2 were located 296 and 157 bp, respectively, upstream of the start of the 16S rRNA gene. Between P2 and the start of the 16S rRNA gene, sequences were identified with typical anti-termination motifs characteristic of E. coli rrn promoter regions. A putative transcription terminator sequence was identified downstream of the 5S rRNA gene and putative primary RNA transcript processing sites at both ends of the lactococcal rRNA operon were also noted.
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Metabolism of polysaccharides by the Streptococcus mutans dexB gene product
More LessSUMMARY: The Streptococcus mutans dexB gene, a member of the multiple sugar metabolism (msm) operon, encodes an intracellular glucan 1,6-α-glucosidase which releases glucose from the non-reducing terminus of α-1,6-linked isomaltosaccharides and dextran. Comparison of primary amino acid sequences showed strong homology to Bacillus oligo-1,6-glucosidases and, like these enzymes, DexB was able to release free glucose from the α-1,4,6-branch point in panose. This suggested a role for DexB in the metabolism of either starch or intracellular polysaccharide, which contain such branch points. However, purified intracellular polysaccharide from the wild-type S. mutans strain LT11 and a mutant deficient in dexB revealed no substantial differences in the extent of branching as demonstrated by iodine staining spectra and the degree of polymerization. Furthermore, thin layer chromatography of radiolabelled intracellular polysaccharide digested with S. mutans wild-type and mutant cell extracts showed no differences in the products obtained. The involvement of DexB in dietary starch metabolism was investigated using α-limit dextrins produced from the action of α-amylase on starch. These induced the msm operon, including dexB, and the DexB enzyme was able to act on the α-limit dextrins to give further fermentable substrates. The transport system encoded by the msm operon can also transport α-limit dextrin. DexB may therefore be important in the metabolism of extracellular starch.
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A new experimental approach to the search for chemical density factors in the regulation of monoculture growth
More LessSUMMARY: In monocultures of micro-organisms, growth is controlled by feedback mechanisms involving chemical factors such as limiting substrates and inhibitory metabolic products. The role of such feedback in the growth regulation of Escherichia coli O-124 was investigated by growing cells in batch culture using a medium containing glucose and mineral salts. In various phases of growth, portions of the native culture were diluted with culture filtrate, so that although cell density decreased, the chemical composition of the growth medium was unaltered. As the diluted cultures grew, variations in growth acceleration were calculated and compared with those of native (undiluted) cultures. Towards the end of the exponential phase and in the growth deceleration phase, the specific feedback level (FBL) was between −20 and −200 (h g 1-1)-1. The feedback components resulting from changes in glucose concentration were calculated using experimentally determined values of μ;max (0.55.0.05 h-1) and K s (2.5 ± 0.7 mg I-1). Only 0.1-40% of FBL could be accounted for by changes in glucose concentration, indicating the presence of additional growth regulators. The method developed may become a new tool for determination of growth-regulating cell-density factors in microbial cultures.
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Temporal activation of β-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool
More LessSUMMARY: β-Glucanase synthesis was temporally activated in Bacillus subtilis at the onset of stationary phase. This regulation was dependent upon a drop in the GTP concentration in response to nutrient limitation. The Spo0A and AbrB proteins were involved in the GTP-dependent temporal activation.
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Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels
More LessSUMMARY: In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase. One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS. In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 °C (permissive) to 36 °C (restrictive). In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions. Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1. A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest. In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift. The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes. Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.
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Influence of medium components and metabolic inhibitors on citric acid production by Penicillium simplicissimum
More LessSUMMARY: Penicillium simplicissimum excreted more than 100 mmol citric acid l-1 [2-9 mmol (g dry wt)-1; 9 d] if an industrial filter dust (> 50 % ZnO) providing a high extracellular buffering capacity was present in the medium. A similar specific [2 mmol (g dry wt)-1], but lower absolute (26 mmol l-1), citric acid excretion occurred in the absence of an extracellular buffer and if amino acids or urea were used as nitrogen source. P. simplicissimum excreted no citric acid under conditions where Aspergillus niger produces citric acid (deficiency of trace elements, low pH and reduced biomass formation). Citric acid excretion by P. simplicissimum always paralleled biomass formation and occurred in a pH range between 4 and 7. This indicated that different imbalances of metabolism were responsible for citric acid excretion in A. niger and P. simplicissimum. However, provided a high extracellular buffering capacity was present, the response of the Penicillium system to different carbon and nitrogen sources was similar to the Aspergillus system. In contrast, the metals iron and copper had virtually no effect on citric acid excretion compared with A. niger. Estimation of intracellular citric acid, as well as the effects of the uncoupler 2,4-dinitrophenol, and the H+-ATPase inhibitor sodium orthovanadate, led to the conclusion that the buffer-stimulated citric acid efflux was dependent on metabolic energy and an energized plasma membrane, respectively. Despite similarities to the Aspergillus system, a different mechanism for buffer-stimulated citric acid excretion by P. simplicissimum seems probable.
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Phosphonate inhibition as a function of phosphate concentration in isolates of Phytophthora palmivora
More LessSUMMARY: Three isolates of Phytophthora palmivora showing differing sensitivities to phosphonate were grown in the presence of a uniform concentration of the anion (1 mM), together with concentrations of phosphate which varied from zero to 3 mM. The phosphonate-sensitive isolate was inhibited by phosphonate at all levels of phosphate, but the resistant isolates were inhibited by phosphonate only when phosphate was limiting to growth and were able to exclude phosphonate more effectively than the sensitive isolate at higher concentrations of phosphate. The percentage inhibition in each strain was proportional to the internal concentration of phosphonate, but the slope of the plot of inhibition against internal phosphonate varied between strains. Differences in the capacity to discriminate between phosphate and phosphonate therefore provide part of, but not the whole explanation for differences in sensitivity between isolates. There was a significant increase in the internal concentration of orthophosphate in the mycelia which were inhibited by phosphonate.
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Differentiation of Frankia strains by their electrophoretic patterns of intracellular esterases and aminopeptidases
More LessSUMMARY: The closely related Frankia strains BR, S21 and Thr, isolated from the genus Casuarina, Allo2 and Dec from the genus Allocasuarina, and G80 from the genus Gymnostoma, could be distinguished by their esterase zymograms after ultra-low gelling point agarose-polyacrylamide electrophoresis. The kanamycin-resistant derivatives S21-kR and BR-kR could also be differentiated from their kanamycin-sensitive parental strains. Different patterns of intracellular esterases could be obtained by using β-naphthyl propionate or 3-indoxyl acetate as substrates. The substrate 5-bromo-4-chloro-3-indoxyl acetate proved particularly useful, revealing a variety of esterases from Gymnostoma isolates. Zymograms of aminopeptidases from all strains were found to be quite similar. Nevertheless, they allowed differentiation of Frankia strains BR and S21 from their kanamycin-resistant derivatives. Aminopeptidase and esterase zymograms obtained from Gymnostoma isolates were markedly different from all others. Zymograms of esterases and aminopeptidases may prove useful for the identification of some other Frankia strains.
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Sodium-stimulated transport of glutamate by Thermus thermophilus strain B
More LessSUMMARY: Thermus thermophilus B is one of the many Thermus strains able to utilize glutamate as a sole source of carbon. Sodium is an obligate requirement for glutamate transport by washed whole cells, but the affinity for sodium (K m = 23 mM) is low. At pH 7.6, uptake of glutamate is rapid in 50 mM-sodium at 65 °C and obeys saturation kinetics with an apparent K m of 0.23 μ;M-glutamate and a V max of 12 nmol glutamate min-1 (mg protein)-1. The transport system is insensitive to osmotic shock and is specific for glutamate, with both the L- and D-isomers being transported. Uptake is very sensitive to inhibitors that collapse the membrane potential (Δψ) or the chemical gradient of sodium ions (ΔpNa), but a transmembrane pH gradient (ΔpH) plays no role in the transport of glutamate. These results are therefore consistent with a membrane sodium/glutamate symport system.
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Effect of short-term variation in irradiance on light harvesting and photosynthesis of the marine diatom Skeletonema costatum: a laboratory study simulating vertical mixing
More LessSUMMARY: A laboratory study was conducted into the physiology of Skeletonema costatum grown under a simple sinusoidal and a fluctuating light regime. The latter simulated a light regime similar to that which could result from the vertical mixing caused by Langmuir circulation. It was shown that the culture simulating vertical mixing reacted by decreasing the photosynthetic unit (PSU) size and increasing the number of PSUs, and hence optimized the rate of maximal photosynthesis at high, saturating irradiances. This culture also showed some change in photosynthetic parameters during the light period, which was especially pronounced during the shift from a low to a higher irradiance. The effect of this on estimates of primary production in a water column is discussed. Further, it is speculated that the assimilation number is regulated by the maximum light intensity experienced during the day rather than the total daily light dose, because only the culture submitted to a fluctuating light regime showed a real change in the maximum rate of photosynthesis (P B max) upon transfer to higher light levels.
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- Plant-Microbe Interactions
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Pilus-mediated adsorption of Pseudomonas syringae to the surface of host and non-host plant leaves
More LessSUMMARY: Adsorption of cells of a variety of Pseudomonas syringae pathovars to the leaf surface of host and non-host plants was measured. The strains used were sensitive to the pilus-specific bacteriophage ø6. Phage-resistant non-piliated mutants were isolated and found to have reduced ability to adsorb to plant surfaces. The pilus-mediated adsorption was not host-specific. Piliated strains adsorbed well to both host and non-host plants. Scanning electron microscopy of the P. syringae pathovar syringae strain R32 and the pathovar phaseolicola strain HB10Y revealed a difference between these strains in the distribution of the bacterial cells over the lower bean leaf surface. P. syringae pv. syringae spread evenly over the leaf surface whereas P. syringae pv. phaseolicola adsorbed preferentially to the stomata. No such localization was observed on chloroform-treated leaves, where the cells of both pathovars were evenly distributed. Adsorption of bacteria to leaf disks was independent of divalent cations, and no specific ionic conditions were required.
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The polar flagellum mediates Azospirillum brasilense adsorption to wheat roots
More LessSUMMARY: Azospirillum brasilense in a motile Gram-negative bacterium that can adapt its flagellation to different environments. Cells growing in a liquid culture possess only a single polar flagellum; growth on a solid surface additionally induces multiple lateral flagella. The polar flagellum is primarily used for swimming, i.e. locomotion of the bacterium in a liquid environment, whereas the lateral flagella allow the bacteria to swarm over a solid surface. We have previously described a completely non-motile A. brasilense mutant (Sp7 p90D084), and shown that this mutant has a drastically reduced adsorption capacity to wheat roots. In the present work, we present several lines of evidence demonstrating that adsorption to wheat roots is mediated by the polar flagellum of A. brasilense. First, the non-adsorbing mutant Sp7 p90D084 forms no polar and no lateral flagella, but is otherwise undistinguishable from wild-type A. brasilense. Second, disintegration of the flagella by heat or acid eliminates adsorption. Third, using a polyclonal antiserum against the polar flagellum filament protein (Fla1), we have isolated out of a collection of 3000 Tn5-B30-induced mutants, three additional and genetically different non-flagellate mutants. Like Sp7 p90D084, these mutants show a severely reduced adsorption capacity to wheat roots. Finally, purified polar flagella bind to wheat roots in vitro.
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- Systematics
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Analysis of intra-specific variation in the fatty acid profiles of Borrelia burgdorferi
More LessSUMMARY: Analysis of the fatty acid methyl esters (FAMEs) of bacteria is a commonly used chemotaxonomic technique. Application of this methodology to spirochaetes associated with Lyme borreliosis revealed distinct clusters corresponding to three genetically distinguished groups: Borrelia burgdorferi sensu stricto, B. garinii, and the VS461 group. However, B. garinii formed a common group with B. hermsii, a relapsing fever spirochaete, and VS461 grouped with B. turicatae and B. parkeri, two other relapsing fever spirochaetes. The diversity in fatty acid profiles of Lyme disease spirochaetes has implications for the protean clinical manifestations of the disease.
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A simple chemical test to distinguish mycobacteria from other mycolic-acid-containing actinomycetes
More LessSUMMARY: Two hundred and fifty-two representatives of the genera Corynebacterium, Gordona, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella were degraded by alkaline hydrolysis and their mycolic acids extracted as methyl esters following phase-transfer-catalysed esterification. When the mycolic acid methyl esters were treated with a mixture of acetonitrile and toluene all mycobacterial mycolates formed copious white precipitates whereas all but 5 out of the 106 non-mycobacterial mycolates remained in solution. The precipitated methyl mycolates and the dried soluble mycolates were compared by pyrolysis gas chromatography and silica gel thin-layer chromatography. On pyrolysis, the precipitated methyl mycolates from mycobacteria yielded fatty acid methyl esters with 20 to 26 carbon atoms whereas those from the remaining taxa produced shorter-chain esters. Mycobacteria and Tsukamurella paurometabola gave multispot mycolic acid patterns on thin-layer chromatography of their methyl esters whereas those from the remaining strains gave single spots. Our results indicate that Rhodococcus chlorophenolicus strains contain mycolic acids typical of mycobacteria. It can be concluded that the mycolic acid precipitation test provides a simple and reliable way of distinguishing mycobacteria from all other prokaryotes, notably from other mycolic-acid-containing taxa.
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Detection of the outer membrane lipoprotein I and its gene in fluorescent and non-fluorescent pseudomonads: implications for taxonomy and diagnosis
More LessSUMMARY: The open reading frame of the OprI lipoprotein gene from Pseudomonas aeruginosa was amplified by polymerase chain reaction (PCR) starting from purified DNA or colony lysates. A fragment of the expected size (249 bp) was detected in all P. aeruginosa strains from various clinical and geographical origins. The gene could only be amplified in pseudomonads of rRNA group I which are considered to be the authentic genus Pseudomonas. Digestions with HaeIII, PvuII and SphI of the amplified fragments demonstrated a sequence variation in the oprI gene. Colony, dot and Western blots with two monoclonal antibodies (mAbs) against the lipoprotein I confirmed our PCR results. These findings open interesting perspectives for the molecular taxonomy of the genus Pseudomonas and the development of diagnostic tools.
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