bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of fungal parasites on cereal cyst nematode (Heterodera avenae Woll.) from naturally infested soil—a scanning electron microscopy study
Authors: Lopez-Llorca L. V. , Duncan G. H.
Published: May 2010

The effects of fungal endoparasites, attacking the eggs of the cereal cyst nematode Heterodera avenae, and details of the infection process were studied by low-temperature scanning electron microscopy. Some female nematodes, even young ones containing no eggs, were colonized by fungi. Spores and hyphae similar to those of Nematophthora gynophila were found in infected specimens. Fungi colonized both roots and nematodes. In early stages of infection, fungi developed within the female nematode between the organs, presumably using the female's body as a food source. In some immature females, the fungi appeared to have destroyed the uterus. In old females, appressoria of Verticillium spp., including V. chlamydosporium, penetrated the eggs they contained and progressively destroyed their contents until the egg shell was filled with hyphae and spores. Only rarely were second-stage juveniles within eggs infected by these fungi. Fungal infection of eggs, which arrests their development at an early stage, therefore occurs predominantly in females rather than in newly formed cysts. Key words: Heterodero avenae, fungal parasites, naturally infested soil, scanning electron microscopy.
p-Hydroxybenzoylformic acid and (R)-(−)-p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolithus arhizus
Authors: Kope H. H. , Tsantrizos Y. S. , Fortin J. A. , Ogilvie K. K.
Published: May 2010

Two antifungal compounds isolated from the liquid culture medium of Pisolithus arhizus were identified as p-hydroxybenzoylformic acid and (R)-(−)-p-hydroxymandelic acid and given the trivial names pisolithin A and pisolithin B, respectively. The efficacy of the compounds to inhibit the germination of conidia of Truncatella hartigii was compared with that of commercially available structural analogues, and a comparable range of effectiveness for 50% germination inhibition (GI50) of conidia was recorded. The commercially available synthetic compounds (R)-mandelic acid, benzoylformic acid, and racemic p-hydroxymandelic acid, had GI50 values of 82, 72, and 59 μg/mL, respectively, as compared with the natural compounds pisolithin A, 67 μg/mL, and pisolithin B, 71 μg/mL. Two synthetic S enantiomers of mandelic acid, (S)-mandelic acid and (S)-(+)-p-hydroxymandelic acid, were the most effective compounds, with GI50 values of 31 and 33 μg/mL, respectively. A sodium salt of mandelic acid had no activity below 500 μg/mL. Pisolithin A and pisolithin B were compared with polyoxin D for inhibition of hyphal growth, as measured by protein estimation. Both pisolithin A and B measured higher levels of putative extractable protein than polyoxin D, but less mycelial wet weight was measured. It is suggested that the pisolithins caused a disruption of cell turgor. A measurement of mycelial dry weights of phytopathogens, incubated with the commercially available analogues, benzoylformic acid and racemic p-hydroxymandelic acid, indicated that benzoylformic acid was either more effective than, or as effective as, racemic p-hydroxymandelic acid or nystatin in arresting fungal growth. For ectomycorrhizal fungi confronted with the pisolithins, both compounds were effective in inhibiting the growth of an equal number of fungi, but benzoylformic acid caused a growth stimulation of some ectomycorrhizal fungi. The result of growth stimulation by the compound is discussed. Key words: Pisolithus, antifungal, ectomycorrhizal, biological control.
X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio κ phage
Authors: Bhattacharyya S. C. , Samad S. A. , Mandal J. C. , Chatterjee S. N.
Published: May 2010

Vibrio cholerae lysogenic κ phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated κ phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m−2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D) – survival (S) curves could be empirically described by the equation S = exp[−(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained. Key words: Vibrio cholerae, lysogenic κ phage, X-ray inactivation, Weigle reactivation, Weigle mutagenesis.
Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages
Authors: Benedi Vicente J. , Regué Miguel , Albertí Sebastián , Camprubí Silvia , Tomás Juan M.
Published: May 2010

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage. Key words: Klebsiella pneumoniae, bacteriophages, isolation, environmental conditions.
Biosynthesis of isochorismate in Klebsiella pneumoniae: origin of O-2
Authors: Zamir Lolita O. , Devor Kenneth A. , Jensen Roy A. , Tiberio Robert , Sauriol Françoise , Mamer Orval
Published: May 2010

The shikimate metabolites are key precursors to a large number of natural products, including aromatic amino acids. Chorismic acid is an important branch point in the biosynthetic pathway to aromatic amino acids. Chorismic acid is also unique among natural products since it is the only compound known to undergo an enzymatic Claisen rearrangement. A metabolite of chorismic acid, isochorismic acid, first observed in Aerobacter aerogenes differs in its chemical structure by the location of the hydroxyl group and the double bonds. Isochorismic acid is a precursor to a growing number of shikimate-derived metabolites. Isochorismic acid has also been postulated to be an intermediate of m-carboxyaromatic amino acids, implying another enzymatic Claisen rearrangement. In this publication, we have isolated isochorismate synthase and found that on lyophilization the enzyme is stable for at least 6 months at −20 °C. Incubation of chorismate with this preparation in water enriched with 18O led to incorporation of one atom of 18O as proven from the fast atom bombardment mass spectra of the HPLC purified derived isochorismate. Key words: chorismate, isochorismate, rearrangement, H218O, isochorismate synthetase.
Effects of bacterial lignin peroxidase on organic carbon mineralization in soil, using recombinant Streptomyces strains
Authors: Wang Zemin , Crawford Don L. , Magnuson Timothy S. , Bleakley Bruce H. , Hertel Greg
Published: May 2010

To study the effects of bacterial lignin peroxidase ALip-P3 of Streptomyces viridosporus T7A on the rate of organic carbon turnover in soil, purified lignin peroxidase, with and without addition of H2O2, was added to sterile and nonsterile silt loam soil. Recombinant Streptomyces lividans strains expressing plasmid-encoded ALip-P3 were also inoculated into the soil. Carbon mineralization was monitored by measuring the rate of CO2 evolution from the soil. In sterile soil, lignin peroxidase addition altered carbon turnover, by increasing the CO2 evolution rate above the near-zero rate of sterile, uninoculated soil. H2O2, when added alone, had no effect, and its addition in combination with peroxidase gave results similar to peroxidase alone. This effect was also observed upon addition of lignin peroxidase to sterile soil already inoculated with S. viridosporus T7A. The increases in soil CO2 evolution rates were also observed in experiments using nonsterile soil. However, results showed more variation, and the effect was shorter lived as a result of lessened peroxidase stability. Three recombinant S. lividans strains expressing the ALip-P3 gene in plasmid pIJ702.LP were also inoculated into soil. There were no significant differences in CO2 evolution rates for sterile soil inoculated with recombinants as compared with sterile soil inoculated with wild-type S. lividans strains. However, in nonsterile soil, addition of the recombinants caused a significantly greater increase in the CO2 evolution rate as compared with the corresponding wild types or S. viridosporus T7A. The effect was short lived, lasting about 5 days. Both the recombinant and wild-type Streptomyces survived in the soil for at least 30 days, and pIJ702.LP was stable in the recombinants in soil. Plasmid pIJ702.LP was transformed into three mutants of S. viridosporus T7A that lacked lignin peroxidase. Plasmid-expressing transformants regained the ability to produce lignin peroxidase. The results show that addition of lignin peroxidase ALip-P3 to soil transiently enhanced the short-term rate of carbon mineralization in the soil. The enhancement was lignin peroxidase specific, since substitution of horseradish peroxidase for lignin peroxidase in the soil addition studies resulted in no enhancement of CO2 evolution. In addition, pIJ702.LP-expressing S. lividans strains also caused the effect, which was significant only in nonsterile soil. Thus, lignin peroxidase ALip-P3 appears to affect the short-term turnover rate of lignin-derived organic carbon in soil, and normal, low lignin peroxidase concentrations in soil may limit the initial turnover rate of lignified plant residues in soil. This is the first report of a genetically engineered microorganism having a measurable effect on a biogeochemical cycle in soil. Key words: Streptomyces, recombinant, lignin, peroxidase, soil.
Antigenic and immunogenic activity of flagella and fimbriae preparations from uropathogenic Proteus mirabilis
Authors: Legnani-Fajardo C. , Zunino P. , Algorta G. , Laborde H. F.
Published: May 2010

The antigenic and immunogenic activities of fimbriae and flagella from three uropathogenic strains of Proteus mirabilis were compared. Flagella were obtained by mechanical treatment and fimbriae were isolated from cells by heat shock, ammonium sulfate precipitation, sodium deoxycholate and urea treatment, and gel filtration. Both preparations inoculated to mice demonstrated high antigenicity. Titers up to 1: 80 000 were determined by an enzyme-linked immunosorbent assay either against the homologous or heterologous strains. When immunized mice were challenged with homologous or heterologous hematogenous infecting doses, a good cross protection was achieved only when fimbriae were used as antigens. Cross-reactivity found between the three fimbriae antisera, and the presence of common proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of fimbriae, should validate the study of these proteins to determine the existence of a shared adhesin. Key words: fimbriae, flagella, Proteus mirabilis, and antigenicity.
Relationship of binucleate Rhizoctonia isolates used for biocontrol of rhizoctonia crown rot of sugar beet to anastomosis systems
Authors: Herr Leonard J.
Published: May 2010

The relationships of 10 binucleate Rhizoctonia isolates used as biocontrol agents of rhizoctonia crown and root rot of sugar beet in Ohio to described binucleate Rhizoctonia anastomosis systems were investigated. Ten Ohio binucleate Rhizoctonia (Ohio BNR) isolates, paired in all combinations, cross anastomosed with one another, indicating that all belong to the same anastomosis group. Four representative Ohio BNR isolates failed to anastomose with any tester isolates of the Ceratobasidium anastomosis grouping system, indicating that none belong in that system. However, all 10 Ohio BNR isolates anastomosed with an AG-B (o) tester isolate (binucleate Rhizoctonia anastomosis grouping system), indicating that the Ohio agents belong in this anastomosis grouping system and to the (o) intraspecific group of AG-B. None of the Ohio BNR isolates anastomosed with either of the other two intraspecific group tester isolates (AG-Ba, AG-Bb) of the AG-B group. Moreover, the AG-B intraspecific group tester isolates, AG-Ba, AG-Bb, AG-B (o), self-anastomosed but did not cross anastomose with one another. Variations in cultural characteristics noted among the 10 Ohio BNR isolates indicated that considerable heterogeneity exists within these AG-B (o) isolates. Key words: binucleate Rhizoctonia, anastomosis, rhizoctonia crown rot, sugar beet.
Polyamine synthetic ability of the eubacteria Halococcus acetoinfaciens, Halococcus agglomeratus, and Halococcus nondenitrificans
Authors: Hamana Koei , Matsuzaki Shigeru
Published: May 2010

Polyamine synthetic activity was compared among the three eubacteria Halococcus acetoinfaciens (IAM 12094), Halococcus agglomeratus (IAM 12095), and Halococcus nondenitrificans (IAM 12096). Putrescine was produced by decarboxylation of ornithine but not from agmatine in the three species. N-Carbamylputrescine was converted to putrescine in the latter two species. Lysine decarboxylase activity was found only in H. acetoinfaciens, which contains aminopropylcadaverine, aminopentylnorspermidine, and N,N′-bis(3-aminopropyl)cadaverine in addition to spermidine, spermine, and thermospermine. In H. acetoinfaciens when diaminopropane was added to the culture medium, both norspermidine and norspermine were synthesized; when homospermidine was added, aminopropylhomospermidine was formed. In contrast H. agglomeratus and H. nondenitrificans synthesize putrescine, spermidine, and trace amounts of spermine but cannot synthesize other triamines and tetraamines from diamines and triamines supplemented to the culture medium. Key words: Halococcus, decarboxylase, polyamines, aminopropyltransferase, inhibitors, aminopropylcadaverine.
Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum
Authors: Park Wan Soo , Murphy Patricia A. , Glatz Bonita A.
Published: May 2010

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3′-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism. Key words: lipid, peroxisomes, oleaginous, Apiotrichum curvatum, catalase, beta-oxidation, glyoxylate cycle.
Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite
Authors: Manocha M. S. , Chen Y.
Published: May 2010

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.
Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus
Authors: Talbot James A. , Nielsen Klaus , Corbeil Lynette B.
Published: May 2010

Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, α2-macro-globulin, but not α1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host–parasite interactions. Key words: cysteine proteinase, Tritrichomonas foetus, host defense proteins, reproductive secretions.
Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis
Authors: Ichiman Yoshitoshi , Usui Yukio , Suganuma Masaru , Yoshida Kosaku
Published: May 2010

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen. Key words: Staphylococcus epidermidis, cell surface antigen, monoclonal IgM antibody, protection.
Simultaneous degradation of acetonitrile and biphenyl by Pseudomonas aeruginosa
Authors: Nawaz Mohamed S. , Chapatwala Kirit D.
Published: May 2010

A bacterium capable of utilizing either acetonitrile as the sole source of carbon and nitrogen or biphenyl as the sole source of carbon was isolated from soil and identified as Pseudomonas aeruginosa. The bacterium also utilized other nitriles, amides, and polychlorinated biphenyls (PCBs) as growth substrates. Acetonitrile- or biphenyl-grown cells oxidized these substrates without a lag. In studies with [14C]acetonitrile, nearly 74% of the carbon was recovered as 14CO2 and 8% was associated with the biomass. In studies with [14C]biphenyl, nearly 68% of the carbon was recovered as 14CO2 and nearly 6% was associated with the biomass. Although higher concentrations of acetonitrile as the sole sources of nitrogen inhibited the rates of [14C]biphenyl mineralization, lower concentrations (0.05%, w/v) gave a 77% stimulation in 14CO2 recovery. Pseudomonas aeruginosa metabolized acetonitrile to ammonia and acetic acid and biphenyl to benzoic acid. The bacterium also simultaneously utilized biphenyl as the sole carbon source and acetonitrile as the sole nitrogen source. However, biphenyl utilization increased only after the depletion of acetronitrile. Metabolites of the mixed substrate were ammonia and benzoic acid, which completely disappeared in the later stages of incubation. Nitrile hydratase and amidase were resposible for the transformation of acetonitrile to acetic acid and ammonia. Key words: biodegradation, acetonitrile, biphenyl, Pseudomonas aeruginosa.
Cloning and characterization of the nifA gene from Herbaspirillum seropedicae strain Z78
Authors: Souza Emanuel M. , Funayama Shigehiro , Rigo Liu U. , Pedrosa Fábio O.
Published: May 2010

A genomic library of Herbaspirillum seropedicae was constructed and screened for the nifA gene by complementation of a nifA mutant of Azospirillum brasilense (FP10). A recombinant plasmid, pEMS1, capable of restoring acetylene reduction activity in the mutant FP10, was isolated and found to hybridize to the nifA gene of Klebsiella pneumoniae. The results suggest that nifA is involved in the regulation of nif genes in H. seropedicae. Key words: diazotroph, nitrogen fixation gene, nif regulation, complementation cloning, Herbaspirillum seropedicae.
Effect of β-glucosidase inhibitors on synthesis of cellulase and β-glucosidase in Sporotrichum (Chrysosporium) thermophile
Authors: Carrel François L. Y. , Canevascini Giorgio
Published: May 2010

The addition of nojirimycin or gluconolactam, substances known as β-glucosidase inhibitors, to cultures of Sporotrichum (Chrysosporium) thermophile growing on cellobiose, caused a twofold or even higher increase (inhibited by cycloheximide) of the intracellular activity of this enzyme, but did not stimulate the synthesis of endocellulase or of cellobiose dehydrogenase. At concentrations from 0.1 to 1.0 mM, nojirimycin stimulated β-glucosidase synthesis to an extent apparently independent of concentration but did not affect cellular respiration on cellobiose. At 5 mM, however, cellobiose assimilation and subsequent growth were significantly but only temporarily impaired and β-glucosidase synthesis, which was still stimulated, was correspondingly delayed. Growth on glucose, on the other hand, was completely unaffected by nojirimycin. Upon fast protein liquid chromatography (FPLC) fractionation by gel filtration of mycelial extracts from cultures grown in the presence and in the absence of nojirimycin (0.1 mM), the β-glucosidase was recovered (67–70%) in two distinct fractions, A and B. The observed increase in β-glucosidase activity for cultures with nojirimycin was found to parallel the increase of fraction B. Key words: β-glucosidase inhibition, nojirimycin, cellulase biosynthesis.
Electrotransformation in Salmonella typhimurium LT2
Authors: Binotto Julie , MacLachlan P. Ronald , Sanderson Kenneth E.
Published: May 2010

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 108–109 electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 106 transformants/μg DNA with strains with Rc or Rd2 LPS, 104 transformants with most smooth and rough strains, and 102 transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhïmurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier. Key words: electroporation, Salmonella, genetics, transformation.
Recombinant interleukin-6 increases the intracellular and extracellular growth of Mycobacterium avium
Authors: Denis Michel , Gregg Evan O.
Published: May 2010

Human monocytes were isolated from the peripheral blood of normal donors and allowed to differentiate in vitro into macrophages. The susceptibility of these cells to infection with a virulent Mycobacterium avium and its modulation by some soluble factors was monitored. The virulent strain of Mycobacterium avium grew progressively in untreated macrophage monolayers. Interleukin-6 (IL-6) was tested for its ability to modulate the macrophage–mycobacteria interaction. Surprisingly, IL-6 was shown to increase M. avium growth in macrophage monolayers by twofold as compared with untreated cells, when added before or after infection. Moreover, addition of rIL-6 to replicating mycobacteria in vitro enhanced their growth two- to three-fold as compared with cultures treated with rIL-6 and a rabbit antiserum to rIL-6. Treatment with IL-6 and interferon-gamma (IFN-γ) or IL-4 did not modify the growth promoting effect of IL-6 in human macrophages. Overall, our results suggest that IL-6 may contribute significantly to the pathogenesis of infections with M. avium by promoting mycobacterial growth. Key words: Mycobacterium avium, macrophages, interleukin-6, interferon-gamma.
Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose
Authors: Cheng K.-J. , Kudo H. , Duncan S. H. , Mesbah A. , Stewart C. S. , Bernalier A. , Fonty G. , Costerton J. W.
Published: May 2010

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi. Key words: cellulolysis, anaerobic rumen fungi, attachment inhibition, digestion inhibition.
Effects of combined treatments of zinc and pH on attached bacterial abundance and activity
Authors: Dean-Ross Deborah
Published: May 2010

The response of attached bacteria to Zn at three pH levels was studied in outdoor stream mesocosms. Streams containing water from Crooked Lake, Indiana, were dosed with either 0.1 or 0.5 mg Zn/L at ambient pH (pH 8.4), pH 7.0, or pH 5.5. Samples were taken at 2, 5, 10, and 20 days and analyzed for total biomass, chlorophyll content, total bacterial numbers, and heterotrophic activity. All measures of community abundance and activity were highest in the treatment receiving 0.1 mg/L at a pH of 8.4. Treatments at lower pH levels and the higher Zn concentration showed significantly lower biomass, chlorophyll content, and total bacterial numbers. Bacterial heterotrophic activity did not demonstrate a statistically significant response as pH decreased, although it did show a statistically significant decrease at a concentration of 0.5 mg Zn/L. The portion of the bacterial community able to grow on nutrient agar demonstrated adaptation to the increased toxicity of Zn at lower pH levels by an increase in the proportion of bacteria resistant to Zn. In addition, the structure of the culturable bacterial community was altered at low pH, as demonstrated by a reduction in the Shannon–Wiener diversity index. The results indicate that pH can modulate Zn toxicity to the attached community. Key words: attached bacteria, zinc, heavy metal resistance, acid rain, species diversity.
Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment
Authors: Belliveau B. H. , Starodub M. E. , Trevors J. T.
Published: May 2010

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains. Key words: Bacillus, plasmid, antibiotic resistance, metal resistance, marine sediment.
Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli
Authors: Hu Y. J. , Smith D. C. , Cheng K. -J. , Forsberg C. W.
Published: May 2010

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, λgtWESλB, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated λFSX101 and λFSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from λFSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-β-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the β-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of β-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan. Key words: rumen bacterium, xylanase gene, λgtWESλB, cellulolysis, Fibrobacter succinogenes.
Immunological studies on staphylococcal enterotoxin D: production of murine monoclonal antibodies and immunopurification
Authors: Shinagawa Kunihiro , Omoe Katsuhiko , Matsusaka Naonori , Sugii Shunji
Published: May 2010

Eight murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin D (SED) were obtained by fusion of myeloma cells with mouse spleen cells immunized with SED only or a combination of SED and either enterotoxin A (SEA) or enterotoxin E (SEE). When only SED was used as an immunogen, six MAbs were specific for SED only, whereas one MAb was reactive with both SED and SEE when both SEs were used as immunogens. One MAb reacted with SEA, SED, and SEE when both SEA and SED were used as immunogens. A MAb with the highest reactivity to SED was used to prepare an immunosorbent for purification of SED by immunoaffinity chromatography. Approximately 70% of the partially purified SED was recovered in the eluate. The purified SED was electrophoretically and antigenically pure. Immunoaffinity chromatography proved useful in the purification of SED in terms of ease of purification, percent enterotoxin, and enterotoxin purity. Key words: enterotoxin D, monoclonal antibodies, Staphylococcus aureus.
Surface-exposed antibody-accessible outer membrane proteins of Bordetella pertussis
Authors: Anwar Hosmin
Published: May 2010

The convalescent sera from a patient recovered form whooping cough were used to identify the surface-exposed antibody-accessible outer membrane proteins (OMPs) of Bordetella pertussis. The results indicated that the 69 000 OMP, the 40 000 porin, agglutinogens 2 and 3, and a number of presently unknown OMPs were exposed on the surface. The importance of these surface-exposed antigens in the protection against whooping cough is discussed. Key words: Bordetella pertussis, whooping cough, immune response, surface antigens, outer membrane protein.
Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk
Authors: Marshall Douglas L. , Schmidt Ronald H.
Published: May 2010

Experiments were designed to elucidate a possible physiological mechanism for stimulated growth of Listeria monocytogenes by Pseudomonas fluorescens in milk. The ability of L. monocytogenes to grow in milk under aerobic and microaerophilic environments was compared. Neither environment favored the growth of the organism at 10 °C. Autoclaved whole milk was inoculated with P. fluorescens P26 and preincubated for 3 days at 10 °C followed by inoculation with L. monocytogenes Scott A and further incubation for 8 days at 10 °C. Changes in selected milk components were monitored over the 8-day period. The amount of lactose in the milk was determined, as well as the extent of proteolysis and lipolysis. Both L. monocytogenes and P. fluorescens were able to hydrolyze milk fat but were unable to use lactose. Milk protein was hydrolyzed by P. fluorescens but not by L. monocytogenes. Whole milk partially proteolyzed by treatment with purified protease was inoculated with L. monocytogenes. Results indicated that the growth of L. monocytogenes was stimulated in proteolyzed milk. This is the first report to provide evidence describing a likely mechanism for commensalism between L. monocytogenes and Pseudomonas spp. Key words: Listeria monocytogenes, Pseudomonas fluorescens, milk proteolysis.
Seasonal variation in presence of Vibrio salmonicida and total bacterial counts in Norwegian fish-farm water
Authors: Enger Øivind , Husevåg Bente , Goksøyr Jostein
Published: May 2010

Total bacterial counts and the number of the fish pathogenic bacterium Vibrio salmonicida have been studied in water samples collected twice a month in 12 Norwegian fish farms from October 1988 to June 1989. Total counts were determined by staining with 4′,6-diamidino-2-phenylindole followed by epifluorescence microscopy. Cells of V. salmonicida were enumerated with a fluorescent antibody technique using highly specific monoclonal antibodies. Despite the fact that no outbreak of cold-water vibriosis was reported, V. salmonicida was detected in all 12 farms, in numbers ranging from 12 to 43 bacteria/mL. The number of farms where V. salmonicida was detected was generally highest during the winter. Total bacterial counts in the water samples varied between 4 × 104 and 9 × 105 bacteria/mL and the lowest numbers occurred during the winter period. The total bacterial counts were comparable with counts in water uninfluenced by fish farming. On the basis of our results, and additional information available about cold-water vibriosis caused by the bacterium V. salmonicida, an asymptomatic carrier state of the disease is proposed. Key words: cold-water vibriosis, asymptomatic carrier, fluorescent antibody, fish pathogen.
In-furrow spray as a delivery system for plant growth-promoting rhizobacteria and other rhizosphere-competent bacteria
Authors: Zablotowicz R. M. , Tipping E. M. , Scher F. M. , Ijzerman M. , Kloepper J. W.
Published: May 2010

A series of greenhouse and field experiments were conducted to evaluate aqueous in-furrow spray techniques for inoculating crop plants with cell suspensions of rhizosphere-competent root-colonizing bacteria. Maximum root colonization of soybean or canola roots by strains of Serratia, Pseudomonas, and Bradyrhizobium occurred using log 8 colony-forming units (cfu)/mL in the spray in greenhouse conditions. Field experiments evaluating a dose reponse demonstrated that maximum soybean root colonization by strains of Serratia or Pseudomonas was achieved between log 7 and 8 cfu/mL, while root colonization by a Bacillus strain was not related to cell concentration in the spray suspension. Root colonization greater than log 4.5 cfu/g root fresh weight was achieved for most strains by in-furrow spray application of a suspension of log 8 cfu/mL at a rate of 10 mL/m for canola, soybean, and wheat, while root colonization of corn ranged from a maximum of log 3.4 to no recovery. In-furrow spray may be a useful method for inoculating plants with rhizosphere-competent bacteria for experimental purposes, thereby avoiding interactions of formulation. It may also have some value for commercial delivery of bacteria to agricultural crops when it is compatible with accepted agronomic practices. Key words: rhizosphere, plant growth promoting rhizobacteria, formulation, Pseudomonas, root colonization.
Chlamydospore-like cells of Candida albicans in the gastrointestinal tract of infected, immunocompromised mice
Authors: Cole G. T. , Seshan K. R. , Phaneuf M. , Lynn K. T.
Published: May 2010

We have demonstrated in a previously described murine model of gastrointestinal (GI) and systemic candidiasis that the antifungal angent cilofungin was efficacious in clearing infection of body organs when administered subcutaneously by infusion, but permitted large numbers of Candida albicans in the GI tract to persist. Yeast and hyphae in these animals were associated primarily with the stratified squamous epithelium of the stomach. Administration of immunocompromising drugs (cyclophosphamide plus cortisone acetate) to animals with persistent GI infection resulted in relapse of systemic candidiasis. Histological examination of the gastric mucosa revealed invasive hyphal elements and yeast as well as multiple chlamydospore-like cells. Comparative histochemical and electron-microscopic examinations of these latter cells produced in host tissue and chlamydospores formed in vitro were conducted. The results suggested that similarities in wall and cytoplasmic composition and ultrastructure exist between these in vivo and in vitro produced C. albicans cells. Exposure of C. albicans to cyclophosphamide during in vitro growth resulted in stimulation of chlamydospore production. No significant effect of cortisone acetate on C. albicans morphogenesis was detected. The murine model used in this study permits investigation of the formation of chlamydospore-like cells of C. albicans during early stages of fungal invasion of cyclophosphamide-treated mice, and of the possible influence of these cells on immunological response of the host to persistent candidiasis of the GI tract. Key words: Candida albicans, chlamydospores, gastrointestinal candidiasis, cyclophosphamide.
Purification and properties of an extracellular xylanase from the thermophilic fungus Humicola grisea var. thermoidea
Authors: Monti Rubens , Terenzi Héctor Francisco , Jorge João Atílio
Published: May 2010

Humicola grisea var. thermoidea mycelium grown on xylan as the sole source of carbon produced at least two extracellular xylanases. The main xylanolytic component (form 2; 90% of recovered activity) was purified to homogeneity. The apparent molecular mass of the purified enzyme was estimated to be 23 000 and 25 550 Da by Bio-Gel P-60 filtration and urea–SDS–PAGE, respectively. The purified enzyme was a glycoprotein with 45% carbohydrate content and pH and temperature optima of 5.5 and 70 °C, respectively. The apparent Km and Vmax values determined with larch-wood xylan were 3.3 mg/mL and 229 μmol∙min−1∙mg protein−1, respectively. The enzyme was highly specific for xylan and degraded this substrate to produce xylo-oligosaccharides, suggesting that it is a β-1,4-endoxylanase (EC 3.2.1.8). The minor enzymatic component of H. grisea extracellular xylanase activity (form 1) was partially purified and some of its properties were studied for comparative purposes. The results obtained suggested that the mode of action of xylanases form 1 and 2 on xylan differs. Key words: xylanase, hemicellulase, enzyme purification, endoxylanase, Humicola grisea.
Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil
Authors: Doyle Jack D. , Short Kevin A. , Stotzky Guenther , King Rick J. , Seidler Ramon J. , Olsen Ronald H.
Published: May 2010

Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil. In soil amended with 2,4-dichlorophenoxyacetate (500 μg/g soil) and inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days. In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude. The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103). The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate. In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103). These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil. Key words: genetically engineered microorganisms, soil, ecology, 2,4-dichlorophenoxyacetate, Pseudomonas putida.
Ultrastructure of basidiospore germination and development of intrasporal hyphae in the snow mold, Coprinus psychromorbidus
Authors: Traquair J. A. , Kokko E. G. , Moskaluk E. R.
Published: May 2010

Basidiospores of Coprinus psychromorbidus, produced for the first time in axenic culture, were germinated on potato dextrose agar in slide culture. After incubation for 24 h, all stages of germination, the development of monokaryotic and dikaryotic hyphae, and the invasion of ungerminated basidiospores were observed using standard transmission electron microscopy. Each basidiospore has a complex, layered wall and dense cell contents containing one nucleus and the normal complement of organelles, plus lipids and dense-body vacuoles. The germ tube emerges through an apical pore in the basidiospore wall. Some abnormal ungerminated spores are invaded by adjacent germling hyphae, which enter through the pore and the hilum. Key words: basidiospore, transmission electron microscopie, invasion.
Formation of transconjugants on plating media following in situ conjugation experiments
Authors: Walter Michael V. , Porteous L. Arlene , Fieland Valerie P. , Seidler Ramon J. , Armstrong John L.
Published: May 2010

Conjugation on agar plates following mating trials can cause transconjugant numbers to be overestimated. Numbers of transconjugants detected after incubating donors and recipients together were compared with those observed when donors and recipients were incubated separately and then mixed immediately prior to plating on selective agar. Mating comparisons were conducted using broth, a soil slurry, and nonsterile soil. Nalidixic acid was added to selective agar plates to investigate its use as an inhibitor of plate mating. The number of transconjugants from broth matings did not significantly differ from the number of transconjugants produced by plate mating on selective media lacking nalidixic acid. Addition of nalidixic acid to selective media reduced the number of transconjugants from broth matings by 10-fold and the number of transconjugants from plate mating after incubation in broth by 100-fold. The number of transconjugants detected from mating experiments in soil slurries was significantly greater than the corresponding plate matings (p = 0.0073). Furthermore, the addition of nalidixic acid to selective agar eliminated all plate matings. In nonsterile soil matings, transconjugants were detected immediately after the inoculation of donors and recipients into soil only when nalidixic acid was absent from the medium. Key words: transconjugants, in situ conjugation, gene exchange in soil, plate matings.