bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and cloning of a 37.6-kb plasmid carried byLegionella pneumophilarecovered from patients and hospital water over a 12-year period
Authors: Mansfield Shawn D. , Bezanson Greg. S. , Marrie Thomas J.
Published: February 2010

For 12 years, strains of Legionella pneumophila serogroup 1 harbouring a 37.6-kb (23 MDa) plasmid have predominated among patient and potable water isolates at the Victoria General Hospital, Halifax, N.S. Plasmid DNA recovered from 24 strains isolated between 1983 and 1995 was digested with the restriction endonucleases EcoRI, HindIII, KpnI, PvuII, XbaI, and BamHI. The distribution of cutting sites indicated that the 23-MDa size group had remained essentially unchanged during this period, suggesting the persistence of a single plasmid type. Further fragmentation pattern analysis permitted the construction of a physical map of the prototype 23-MDA plasmid, pLp4269. Double digestion with BamHI–HindIII enabled the cloning of 94.4% of pLp4269 into pBluescript vector. A 2.1-kb fragment was not clonable. Plasmid pLp4269 is the first of the smaller Legionella extrachromosomal DNAs to be characterized in this way.Key words: Legionella, plasmid, stability, map, cloning.
The 2, 4-dichlorophenol hydroxylase ofAlcaligenes eutrophusJMP134 is a homotetramer
Authors: Farhana Lulu , New Peter B.
Published: February 2010

2, 4-Dichlorophenol hydroxylase (DCP-hydroxylase) is a key enzyme in the pathway for degradation of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in many bacteria. In Alcaligenes eutrophus JMP134, DCP-hydroxylase was reported to consist of two dissimilar types of subunit of 66 and 45 kDa, a structure which is different from that in other bacteria. Using a different procedure involving affinity purification and ion-exchange chromatography, we have purified active enzyme from JMP134 and show that it has a native molecular mass of approximately 245 kDa and consists of a single type of subunit of 66 kDa, similar to all other flavoprotein monooxygenase enzymes. A 45-kDa polypeptide, found in partially purified enzyme preparations, was not required for enzyme activity but had some serologic and N-terminal amino acid sequence similarity to the 66-kDa enzyme subunit.Key words: 2, 4-dichlorophenol hydroxylase, Alcaligenes eutrophus, 2, 4-D biodegradation.
Degradation of pyridine by Nocardioides sp. strain OS4 isolated from the oxic zone of a spent shale column
Authors: Rhee Sung-Keun , Lee Sung-Taik , Lee Ki-Young , Chung Jae-Chun
Published: February 2010

A pyridine-degrading bacterial strain was isolated from the oxic zone of a spent shale column. The microorganism was an aerobic and pleomorphic coryneform bacterium with LL-diaminopimelic acid in the cell wall. On the basis of its phylogenetic and chemotaxonomic characteristics, the strain was identified as Nocardioides sp. strain OS4. The pyridine was completely degraded and the growth yield was 0.30 g cell/g pyridine. Strain OS4 metabolized pyridine in an inducible manner and released a pigment that has maximum absorbance at 400 nm during the pyridine degradation. This strain also degraded some compounds of the basic fraction of retort water and various other aromatic compounds.Key words: pyridine, biodegradation, Nocardioides sp., retort water.
Ultrastructure and geochemistry of endolithic microorganisms in limestone of the Niagara Escarpment
Authors: Ferris F. G. , Lowson E. A.
Published: February 2010

Endolithic microbial communities dominated by photosynthetic cyanobacteria occur at a depth of 0.5 – 3.0 mm inside dolomitic limestone that forms the vertical cliffs of the Niagara Escarpment in Ontario, Canada. Coccoid Gloeocapsa was the most prominent cyanobacterial species in colonized rock samples examined by differential interference contrast and episcopic fluorescent microscopy. Filamentous varieties of cyanobacteria were less common than coccoid forms but could be easily distinguished in all slide mount preparations. Scanning electron microscopy showed that the endoliths occurred in communal populations situated within the open pore spaces of the limestone. Heterotrophic bacteria were also observed growing as epiphytes on dead and living cyanobacteria, and in epilithic biofilms on pore space walls. In thin-sectioned specimens examined by transmission electron microscopy, these adherent bacteria commonly exhibited extensive accumulations of fibrous extracellular polymeric materials external to their cell walls. Biomass concentrations, estimated from the organic phosphorus content of the samples, generally paralleled rock porosity and ranged from 3.3 to 17.1% dry weight. Carbon isotope (14C) measurements indicated that atmospheric carbon dioxide is used by the endolithic cyanobacteria, rather than dissolved inorganic carbon from the weathering of carbonate minerals in the limestone host rock. In addition, whole rock multielement analyses revealed an enrichment of some elements (e.g., phosphorus, barium, lead, and zinc) in the endolith zone over the host rock, while other elements (e.g., magnesium, calcium, iron, and copper) were depleted. The implication is that the endolithic microorganisms play an active role in the biogeochemical cycling of nutrient and trace elements in cliff face ecosystems of the Niagara Escarpment.Key words: limestone, endolith, biofilm, cyanobacteria, geochemistry.
Production of an extracellular trypsin-like protease by the fungal plant pathogenVerticillium dahliae
Authors: Dobinson Katherine F. , Lecomte Nicolas , Lazarovits George
Published: February 2010

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate Nα-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.Key words: proteinase, phytopathogen, verticillium wilt, wilt fungi.
Subtyping ofNeisseria meningitidisstrains isolated in Quebec, Canada: correlation between deduced amino acid sequences and serosubtyping techniques
Authors: Arhin Francis F. , Moreau France , Mills Elaine L. , Coulton James W.
Published: February 2010

Routine serosubtyping of Neisseria meningitidis relies upon reactivity of whole cells to monoclonal antibodies (mAbs). This procedure is limited in providing maximum serosubtype information because some epitopes in whole cells are masked and because mAbs are currently unavailable for some epitopes. To address masking of epitopes in whole cells, we isolated outer membrane vesicles (OMVs) from nine representative meningococcal strains that were isolated (1991–1993) in the province of Quebec, Canada; the OMVs were used in enzyme-linked immunosorbent assay for reactivity to mAbs, and improved serosubtyping information was obtained. A recent proposal assigns subtypes based on deduced amino acid sequences in the variable regions of the class 1 outer membrane protein. This scheme maintains the subtyping nomenclature that is based on reactivity to mAbs by defining the sequences in the epitopes recognized by the mAbs. We used this technique to assign subtypes to the meningococcal strains isolated in Quebec. For the strains tested, serosubtyping using mAbs and subtyping based on deduced amino acid sequences were in complete agreement. Subtyping using deduced amino acid sequences is superior because it does not depend on the availability of mAbs.Key words: Neisseria meningitidis, outer membrane proteins, serosubtyping, PorA.
Characterization of microbial communities in deep groundwater from granitic rock
Authors: Jain D. K. , Providenti M. , Tanner C. , Cord I. , Stroes-Gascoyne S.
Published: February 2010

The microbial characteristics of deep granitic nutrient-poor groundwater from two boreholes at the Underground Research Laboratory of Atomic Energy of Canada Limited were studied. Scanning electron microscopy of the groundwater samples revealed significant numbers of bacteria of various sizes and shapes, including spherical, rod, and curved shaped. A few bacteria with appendages were also observed. Significant numbers of bacteria (~105/mL) were enumerated using acridine orange (AO) staining. An active microbial population was detected with three direct methods and it ranged from 1 to 83% of the AO count, depending on the method used. Culturable aerobic and anaerobic (including facultative) heterotrophic bacteria ranged from 0.06 to 10.2% and 0.008 to 7.35%, respectively, of the AO count. Denitrifying, N2-fixing, sulphate-reducing, and iron-precipitating bacteria were present, but no iron-oxidizing bacteria or methanogens could be detected. Tentative identification of 160 isolates using the Biolog® system showed a predominance of three Pseudomonas species, P. fluorescens, P. marginalis, and P. corrugata. Phospholipid fatty acid analysis showed that the bacteria in the groundwater samples faced starvation stress. However, laboratory studies showed that these bacteria can efficiently uptake and mineralize organic substrates when supplied.Key words: deep groundwater, microbial communities, characterization.
Cluster analysis of genotypically characterized Lactobacillus species based on physicochemical cell surface properties and their relationship with adhesion to hexadecane
Authors: Millsap Kevin W. , Reid Gregor , Mei Henny C. van der , Busscher Henk J.
Published: February 2010

Lactobacilli can interfere with the adhesion of uropathogens to uroepithelial cells and catheter materials through a variety of mechanisms, such as adhesion. Lactobacillus adhesion to substratum surfaces has been theorized to result from the physicochemical properties of the interacting surfaces. In this paper physicochemical cell-surface properties, including hydrophobicity (determined by water contact angles), pH dependence of zeta potentials, elemental surface composition (determined by X-ray photoelectron spectroscopy), and adhesion to hexadecane, of four genotypically characterized Lactobacillus species (eight L. acidophilus, eight L. casei, four L. fermentum, and seven L. plantarum strains) were determined to see whether a grouping of the strains according to their phenotypes could be obtained that corresponded with the genotypic characterization of the strains. The strains showed major differences in physicochemical cell-surface properties: at the species level relationships could be observed between water contact angles, isoelectric points, and the N/C and O/C elemental surface concentration ratios, with nitrogen-containing groups (proteins) being responsible for increased hydrophobicities and isoelectric points, and oxygen-containing groups (phosphates and polysaccharides) yielding decreased hydrophobicities and isoelectric points. A hierarchical cluster analysis grouped all L. acidophilus strains in one well-separated cluster that also included two L. casei and two L. fermentum strains. Separation of L. acidophilus from the other species was done predominantly on the basis of increased cell surface hydrophobicity (average water contact angle of 63°) and isoelectric point (approximately pH 3.3) as compared with the other species, which had lower water contact angles and isoelectric points, and corresponded with the observation that only L. acidophilus strains adhered in measurable numbers to hexadecane. Also, the L. plantarum strains were grouped closely together in one cluster, but this cluster was heterogeneous due to the inclusion of L. casei and L. fermentum strains.Key words: Lactobacillus, surface properties, hydrophobicity, zeta potential, adhesion.
Microbial degradation of quadricyclane-derived tricyclo[2.2.1.02,6]heptan-3-ol in soil
Authors: Jin Song , Swoboda-Colberg Norbert G. , Colberg Patricia J. S.
Published: February 2010

Quadricyclane (tetracyclo[3.2.02,7.04,6]heptane) is a strained, saturated cyclic hydrocarbon and a potential environmental contaminant because of uses in solar energy and optical memory storage devices and as a recently proposed additive to jet fuel. Quadricyclane is very unstable in both soil and water, and in the environment it exists as abiotic transformation products, tricyclo[2.2.1.02,6]heptan-3-ol and bicyclo[2.2.1]hept-5-en-2-ol. In soils, tricyclo[2.2.1.02,6]heptan-3-ol is microbially transformed to a C7H8O intermediate which we postulate to be tricyclo[2.2.1.02,6]heptan-3-one. This compound is subsequently biodegraded over several months. Elevated respiration rates in soils amended with tricyclo[2.2.1.02,6]heptan-3-ol provide evidence that it is mineralized to CO2, while bicyclo[2.2.1]hept-5-en-2-ol concentrations in soil microcosms remained unchanged over a period of 8 months.Key words: quadricyclane, biotransformation, biodégradation, tricyclo[2.2.1.02,6]heptan-3-ol, bicyclo[2.2.1]hept-5-en-2-ol.
Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae
Authors: Pinto Alexandre de Siqueira , Barreto Cristine Chaves , Vainstein Marilene Henning , Schrank Augusto , Ulhoa Cirano José
Published: February 2010

Chitinases are produced by Metarhizium anisopliae when it is grown in the presence of chitin. A chitinase from the culture filtrate of Metarhizium anisopliae was successively purified by precipitation with ammonium sulphate, followed by anion-exchange chromatography on DEAE-Sephacel. The purified enzyme, which has a molecular mass of approximately 30 kDa by sodium dodecyl sulphate – polyacrylamide gel electrophoresis, catalyses the hydrolysis of p-nitrophenyl β-N-diacetylchitobiose with an apparent Km of 0.537 mmol and Vmax of 4.86 nmol∙mL−1∙min−1. The optimum pH and temperature were 4.5–5.0 and 40–45 °C, respectively.Key words: chitinases, Metarhizium anisopliae, enzyme purification, enzyme characterization.
Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative
Authors: Mahaffee W. F. , Kloepper J. W.
Published: February 2010

The future use of genetically modified microorganisms in the environment will be dependent on the ability to assess potential or theoretical risks associated with their introduction into natural ecosystems. To assess potential risks, several ecological parameters must be examined, including the impact of the introduced genetically modified organism on the microbial communities associated with the environment into which the introduction will occur. A 2-year field study was established to examine whether the indigenous bacterial communities of the rhizosphere and endorhiza (internal root tissues) were affected differently by the introduction of an unaltered wild type and its genetically modified derivative. Treatments consisted of the wild-type strain Pseudomonas fluorescens 89B-27 and a bioluminescent derivative GEM-8 (89B-27::Tn4431). Cucumber root or seed samples were taken 0, 7, 14, 21, 35, and 70 days after planting (DAP) in 1994 and 0, 7, 14, 28, 42, and 70 DAP in 1995. Samples were processed to examine the bacterial communities of both the rhizosphere and endorhiza. Over 7200 bacterial colonies were isolated from the rhizosphere and endorhiza and identified using the Sherlock System (Microbial ID, Inc.) for fatty acid methyl ester analysis. Community structure at the genus level was assessed using genera richness and Hill's diversity numbers, N1 and N2. The aerobic–heterotrophic bacterial community structure at the genus level did not significantly vary between treatments but did differ temporally. The data indicate that the introduction of the genetically modified derivative of 89B-27 did not pose a greater environmental risk than its unaltered wild type with respect to aerobic–heterotrophic bacterial community structure.Key words: diversity, ecology, PGPR, Pseudomonas, root colonizaton, GEM.
Inoculum density, temperature, and genotype effects on in vitro growth promotion and epiphytic and endophytic colonization of tomato (Lycopersicon esculentumL.) seedlings inoculated with a pseudomonad bacterium
Authors: Pillay V. K. , Nowak J.
Published: February 2010

The effects of inoculum density (0, 4.6 × 107, 4.2 × 108, and 8.8 × 108 cfu∙mL−1), temperature (10, 20, and 30 °C), and plant genotype (cultivars Celebrity, Blazer, Scotia, and Mountain Delight) on bacterial colonization and plant growth promotion were investigated in a gnotobiotic system. An in vitro dual culture of tomato (Lycopersicon esculentum L.) plantlets and a Pseudomonas sp., strain PsJN, were used. Epiphytic (external) and endophytic (internal) bacterial populations were determined to evaluate plantlet colonization. Shoot and root biomass of bacterized plantlets was significantly higher (p ≤ 0.05) than that of nonbacterized controls. Growth promotion was best with inoculum densities of 3 × 108– 7 × 108 cfu∙mL−1at 20 °C, particularly in the early maturing cultivars Blazer and Scotia. Lower inoculum densities were required to maximize root growth (approximately 1 × 108 cfu∙mL−1) than shoot growth (approximately 3 × 108 cfu∙mL−1). Shoot surface populations did not vary with inoculum density or temperature, but the bacterium colonized the shoot exterior of cultivars Celebrity, Mountain Delight, and Scotia better than cultivar Blazer. The root surface populations increased linearly with increasing inoculum density (within a range of 107–108 cfu∙mL−1), decreased with increasing temperatures (from 10 to 30 °C), and were higher for the main season cultivar Celebrity than for cultivars Blazer, Scotia, and Mountain Delight. Populations of shoot endophytes did not vary with initial inoculum density or genotype but were affected by temperature; the highest colonization was at 10 °C. The number of root endophytes was also highest at 10 °C at the inoculum density of approximately 4 × 108 cfu∙mL−1and did not vary with genotypes. The experiments clearly indicate that there was no relationship between root surface colonization and plant growth promotion. However, the range of inoculum levels (3 × 108– 7 × 108 cfu∙mL−1) that promoted colonization of the inner root tissues (endophytic) also best promoted plant growth. A possible biostimulation threshold within the tissues of the inoculated plants under conditions favourable to the growth of tomato is proposed.Key words: Pseudomonas sp., tomato, colonization, growth promotion.
Biodiversity of yeasts from Illinois maize
Authors: Nout M. J. R. , Platis C. E. , Wicklow D. T.
Published: February 2010

Microflora in wound sites of preharvest maize (including bacteria, yeasts, and filamentous fungi) may play a role in attracting insects to maize plants and may also interact with growth and mycotoxin production by filamentous fungi. As little data are available about the yeasts occurring on maize from the U.S. corn belt, samples of milled maize from experimental plantings at the University of Illinois River Valley Sand Field were analyzed. Yeast counts showed slight yearly fluctuation and varied between 3.60 and 5.88 (log cfu/g maize). The majority of the yeasts were Candida guilliermondii (approximately 55%), Candida zeylanoides (24 %), Candida shehatae (11%), and Debaryomyces hansenii (3%). Also present were Trichosporon cutaneum, Cryptococcus albidus var. aerius, and Pichia membranifaciens. The occurrence of killer yeasts was also evaluated. Killer yeasts were detected in maize for the first time and were identified as Trichosporon cutaneum and Candida zeylanoides. These were able to kill some representative yeasts isolated from maize, including Candida guilliermondii, Candida shehatae, and Cryptococcus albidus var. aerius. Other maize yeasts (Candida zeylanoides, Debaryomyces hansenii, Pichia membranifaciens) were not affected. The majority of yeasts found on maize were unable to ferment its major sugars, i.e., sucrose and maltose. Some (e.g., Candida zeylanoides) were not even able to assimilate these sugars. The importance of these properties in relation to insect attraction to preharvest ears of maize is discussed.Key words: corn, maize, yeast, killer.
Comparison of static and agitated immobilized cultures of Phanerochaete chrysosporium for the degradation of pentachlorophenol and its metabolite pentachloroanisole
Authors: Laugero Chantal , Sigoillot Jean-Claude , Moukha Serge , Asther Marcel , Mougin Christian
Published: February 2010

The transformation of pentachlorophenol (PCP) by Phanerochaete chrysosporium I-1512 in relation to pentachloroanisole (PCA) was studied in two different modes of culture. The degradation of PCP by immobilized cells in agitated medium was compared with that by static cultures. Results clearly established the advantage of an immobilized culture for mineralization of PCP: 23% of CO2 released for fungus immobilized on stainless steel mesh rings as compared with 11% for static cultures. PCA, a metabolite of PCP, was formed only in static cultures and underwent a limited mineralization. Moreover, experiments performed with mycelium and culture supernatant (in the presence of cycloheximide) clearly demonstrated that PCA formation was catalyzed by the biomass in static cultures of P. chrysosporium. Assays in vitro did not establish any involvement of lignin or manganese peroxidases in PCP or PCA transformation.Key words: filamentous fungi, Phanerochaete chrysosporium, pentachlorophenol, pentachloroanisole.
Bioengineering of emulsifier structure: emulsan analogs
Authors: Gorkovenko Alexander , Zhang Jinwen , Gross Richard A. , Allen Alfred L. , Kaplan David L.
Published: February 2010

Strategies were investigated to modulate the side chain structure of emulsans formed by Acinetobacter calcoaceticus RAG-1. Analysis of emulsan fatty acid side chain groups by gas chromatography – mass spectrometry (GC–MS) revealed that by providing the exogenous n-alkanoic fatty acids 15:0, 16:0, and 17:0, emulsan analogs were formed with 53, 46, and 44 mol%, respectively, of fatty acid substituents with chain lengths equal to that of the carbon source. In contrast, the increase in emulsan fatty acids of chain lengths less than 15 or greater than 17 by providing corresponding shorter and longer chain length fatty acids as carbon sources was not substantial. When [1-13C]-labeled (99% enriched) palmitic acid was used as a carbon source along with acetate, analysis of m/z 75/14 and 87/88 isotopomer ratios by GC-MS indicated that 84 and 86% of the 16:0 and 16:1 (9-cis) side groups, respectively, were incorporated intact from the 16:0 carbon source. The percentage of 14-, 15-, 16-, 17-, and 18-carbon chain length fatty acid esters that were monounsaturated were 11, 26, 50, 70, and 85%, respectively. Based on the observed percentage of unsaturated chain length dependence and almost identical enrichment at C-1 of 16:0 and 16:1 (9-cis) side groups from [1-13C]-labeled experiments, it was concluded that desaturation of preformed n-alkanoic acids was the predominant mechanism of their formation. Further work established correlations between side chain structure and product emulsification specificity/activity, so that bioengineered emulsans with improved selectivity can now be formed.Key words: emulsan, Acinetobacter calcoaceticus RAG-1, fatty acids, direct incorporation, emulsification activity.
Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin
Authors: Marri Laura , Barboni Emanuela , Irdani Tiziana , Perito Brunella , Mastromei Giorgio
Published: February 2010

Streptomyces rochei A2 endoglucanase (eglS) and β-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgsl probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis of genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.Key words: Streptomyces, cellulase genes, hybridization, restriction enzyme analysis.
Identification of gene loci controlling pectate lyase production and soft-rot pathogenicity in Pseudomonas marginalis
Authors: Liao Ching-Hsing , McCallus Daniel E. , Fett William F. , Kang Yue-gyu
Published: February 2010

Pseudomonas marginalis is an important postharvest pathogen capable of causing soft rot in a wide variety of harvested fruits and vegetables. Following transposon mutagenesis, we isolated two groups of P. marginalis CY091 mutants deficient in production of pectate lyase (Pel) and soft-rot pathogenicity in plants. The first group, designated Pel−, was caused by the insertion of Tn5 into a pel structural gene, and the second group, designated LemA−, was caused by the insertion of Tn5 into a regulatory locus corresponding to the lemA gene previously identified in other Gram-negative bacteria. The LemA− mutants also exhibited alteration in colony morphology and showed deficiency in production of protease (Prt). A cosmid clone pCIC carrying the P. marginalis lemA gene was isolated and characterized. pCIC was capable of restoring Pel production and soft-rot pathogenicity in LemA− mutants of P. marginalis and Pseudomonas viridiflava, indicating that the function of lemA gene in these two pseudomonads was similar and interchangeable. Using MudI-mediated mutagenesis, we isolated a third group of P. marginalis mutants deficient in production of Pel, Prt, and soft-rot pathogenicity. Mutants in this group (designated GacA−1) contained an insertion of MudI in a locus corresponding to the gacA gene of P. viridiflava. Like LemA− mutants, GacA− mutants also exhibited alteration in colony morphology and showed deficiency in production of Pel and Prt. However, GacA− mutants produced much lower levels of levan and fluorescent pyoverdine siderophore than the wild type and LemA− mutants. These results provide the first genetic evidence that P. marginalis produces a single alkaline Pel for maceration of plant tissue and demonstrate that production of Pel, Prt, levan, and pyoverdin by this bacterium is mediated by the two-component lemA/gacA gene system.Key words: two-component regulators, pectate lyase, protease, levan, pyoverdin.
Effect of carbon source on extracellular (1 → 3)- and (1 → 6)-β-glucanase production byAcremonium persicinum
Authors: Pitson Stuart M. , Seviour Robert J. , McDougall Barbara M.
Published: February 2010

The effect of carbon source on the levels of three (1 → 3)-β-glucanases and a (1 → 6)-β-glucanase in the culture filtrates of the filamentous fungus Acremonium persicinum was investigated. All four enzymes were produced during growth of the fungus on (1 → 3)-, (1 → 6)-, and (1 → 3)(1 → 6)-β-glucans as well as β-linked oligoglucosides. However, only one (1 → 3)-β-glucanase and the (1 → 6)-β-glucanase were detected during growth on a range of other carbon sources including glucose, carboxymethylcellulose, and the α-glucan pullulan. The presence of glucose in the medium markedly decreased the production of all four glucanases, although the concentration required to effect complete repression of enzyme levels varied for the different enzymes. Similar repressive effects were also observed with sucrose, fructose, and galactose. The most likely explanations for these observations are that the synthesis of the (1 → 6)-β-glucanase and one of the (1 → 3)-β-glucanases is controlled by carbon catabolite repression, while the remaining two (1 → 3)-β-glucanases are inducible enzymes subject to carbon catabolite repression.Key words: (1 → 3)-β-glucanase, (1 → 6)-β-glucanase, Acremonium persicinum, regulation of synthesis, fungal β-glucanases.
Denitration of 2,4,6-trinitrotoluene byPseudomonas savastanoi
Authors: Martin J. L. , Comfort S. D. , Shea P. J. , Drijber R. A. , Kokjohn T. A.
Published: February 2010

Past disposal of wastewaters containing 2,4,6-trinitrotoluene (TNT) at the former Nebraska Ordnance Plant has resulted in numerous acres of TNT-contaminated soil. Examining the microbial population of these soils revealed several TNT-tolerant Pseudomonas spp. We selected one species, P. savastanoi, to determine its ability to transform TNT. Pure culture experiments were performed in pseudomonas minimal medium containing 0.31 mM TNT (70 mg TNT∙L−1) under varied nutrient and cell density regimes. Experiments with TNT as a sole C or N source showed that P. savastanoi has the ability to denitrate TNT, as evidenced by production of 2,4-dinitrotoluene (2,4-DNT) and NO2−with time. TNT denitration and formation of 2,4-DNT were enhanced by removing NH4+and adding NO2−to the growth medium. In all experiments, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) appeared as incidental reduction products. Glucose addition to the medium enhanced 2-ADNT and 4-ADNT production and decreased denitration of TNT. Mid-log phase cells rapidly transformed [ring-14C(U)]TNT but were unable to mineralize significant quantities of TNT, as evidenced by conversion of less than 1% of the label to14CO2. These results indicate that P. savastanoi is a TNT-tolerant pseudomonad that can promote TNT degradation through reductive denitration and nitro moiety reduction.Key words: TNT, biodegradation, transformation, reduction, nitrite.
Isolation ofAzospirillum lipoferumfrom the rhizosphere of rice by a new, simple method
Authors: Van Vân Trân , Ngôkê Suong , Berge Odile , Hebbar Prakash , Heulin Thierry , Faure Denis , Bally René
Published: February 2010

A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10−1to 10−10of rice root macerates and rhizosphere soil were incubated at 28 °C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 × 103/g dry root weight in KCl, 2 × 104/g dry root weight in H2O, 2 × 106/g dry root weight in malate, and 3 × 106/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing less than 0.001% of the total microflora. The isolates had vibrioid to S-shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their identification as Azospirillum lipoferum was based on biochemical tests and hybridization with a 16S rRNA probe specific for this species and was confirmed by the detection of a 150-MDa plasmid.Key words: rice, rhizosphere, soil extract, isolation, identification, Azospirillum.
Insertion sequence IST3091 of Thiobacillus ferrooxidans
Authors: Chakravarty Leena , Kittle Jr. Joseph D. , Tuovinen Olli H.
Published: February 2010

An insertion sequence, designated as IST3091, was located adjacent to the putative origin of replication region of plasmid pTFI91 of Thiobacillus ferrooxidans TFI-91. The DNA sequence of the transposase gene of IST3091 revealed similarity with that of IS30, IS1086, IS4351, and the integrase gene of SpV1-R8A2 B (a bacteriophage of Spiroplasma citri). The sequence of IST3091 is 1063 bp long with partially matched 30-bp terminal inverted repeats. Several restriction fragments of plasmid pTFI91 of T. ferrooxidans containing the IST3091 element were cloned into the vector pHSG398. The hybrid plasmids (pBTL) were transformed into Escherichia coli NK7379 containing a miniF plasmid, which was devoid of transposable elements. The transposition function of the IST3091 element was confirmed by mobilizing hybrid plasmids via conjugation from transformed E. coli NK7379 (donor) to E. coli M8820 (recipient). The presence of the transposed element in transconjugants was detected by polymerase chain reaction amplification.Key words: insertion element, Thiobacillus ferrooxidans, transformation, transposase.
Increased cellular fatty acid desaturation as a possible key factor in thermotolerance inSaccharomyces cerevisiae
Authors: Guerzoni Maria Elisabetta , Ferruzzi Marilena , Sinigaglia Milena , Criscuoli Gian Carlo
Published: February 2010

An increase of the unsaturation level of the cellular fatty acids was observed at sublethal or superoptimal temperatures in Saccharomyces cerevisiae. The hypothesis of this paper is that a high unsaturated fatty acids relative content "per se" is not a prerequisite for withstanding sublethal temperature stress in yeast but is the result of oxygen-consuming desaturase activation, with consequent reduction of oxygen and the oxygen free radicals as they form during thermal stress. In the thermotolerant strains, no increase of cellular thiobarbituric acid reactive substances (TBARSs) was observed when temperature approached the maximal growth temperature, suggesting prevention of oxidative damage. On the other hand, the values of TBARSs tripled at 42 °C in nonthermotolerant strains. When a sublethal hydrogen peroxide treatment preceded a rapid temperature rise, a selected thermotolerant strain responded with a relative increase of saturated fatty acids. This response, associated with an insignificant viability loss due to the double stress, suggests the induction an alternative oxygen consumption mechanism preventing excessive fatty acid unsaturation, which could be detrimental to the cells in the presence of hydrogen peroxide at sublethal temperatures.Key words: Saccharomyces cerevisiae, fatty acid composition, desaturase, thermotolerance, oxidative stress.
Characteristics of a nitropropanol-metabolizing bacterium isolated from the rumen
Authors: Anderson Robin C. , Rasmussen Mark A. , Allison Milton J. , DiSpirito Alan A.
Published: February 2010

We report some characteristics of a ruminai bacterium (strain NPOH1) that metabolizes 3-nitropropanol, the toxic principle of various milk vetchs that are distributed worldwide. The gram-positive bacterium was nonmotile and did not produce spores. Growth of strain NPOH1 occurred under anaerobic conditions and was supported by the electron acceptors 3-nitropropanol, 3-nitropropionate, nitrate, 2-nitropropanol, nitroethane, nitroethanol, or 3-nitro-1-propyl-β-D-glucopyranoside (miserotoxin). Other potential electron acceptors, namely sulfate, sulfite, azide, chlorate, perchlorate, nitrite, fumarate, 2-nitrobutane, or nitrobenzene, did not support growth. Formate, lactate, and H2stimulated growth of strain NPOH1 in the presence of the appropriate nitrocompound, whereas a variety of other potential H2donors did not. When grown in medium containing both nitrate and either 3-nitropropanol or 3-nitropropionate, nitrate was the preferred acceptor. Strain NPOH1 reduced nitrate to nitrite and, when grown with excess reductant, nitrite was further reduced to ammonia. The products formed during the metabolism of 3-nitropropanol and 3-nitropropionate by mixed ruminal populations, 3-aminopropanol and β-alanine, were not found in culture fluids of strain NPOH1. Analysis of total cellular fatty acid profiles and of the mole percent guanine plus cytosine suggests that strain NPOH1 is a novel bacterium. The capacity of strain NPOH1 to metabolize 3-nitropropanol suggests that this organism may play an important role in detoxification of 3-nitropropanol in the rumen.Key words: nitropropanol, nitropropionate, anaerobic, rumen, detoxification.
The interaction of the soft rot bacterium Pseudomonas gladioli pv. agaricicola with Japanese cultivated mushrooms
Authors: Gill Warwick M. , Tsuneda Akihiko
Published: February 2010

The mushroom soft rot bacterium Pseudomonas gladioli pv. agaricicola was observed to cause pitting when inoculated onto tissues of several commercially important Japanese cultivated mushrooms. Scanning electron microscope studies demonstrated the sequential removal of hyphal wall layers, thereby exposing the chitin skeletal matrix, which in turn was degraded. A second type of damage typified by collapsed, shriveled, and in some cases lysed hyphal cells was also observed. Culture plate assays revealed that Pseudomonas gladioli pv. agaricicola produces chitinase and this, coupled with earlier evidence of a β-glucanase enzyme, accounted for the degradative ability of the pathogen. The gelatinous coating on the Pholiota nameko sporocarp appeared to confer resistance to Pseudomonas gladioli pv. agaricicola attack. Petri dish coincubations with several cultivated mushroom species indicated the ability of Pseudomonas gladioli pv. agaricicola to inhibit mycelial growth over a large distance and suggested the presence of a toxin or toxins. Owing to its wide host range, Pseudomonas gladioli pv. agaricicola is considered as a potential threat, not only to the mushroom industry in Japan but also to the mushroom industry in other tropical/subtropical countries.Key words: chitinase, disease, Pseudomonas gladioli pv. agaricicola, soft rot, toxin.
A rapid viability assay forCryptosporidiumoocysts andGiardiacysts for use in conjunction with indirect fluorescent antibody detection
Authors: Dowd Scot E. , Pillai Suresh D.
Published: February 2010

There is an urgent need to develop rapid methods to determine the viability of Cryptosporidium oocysts and Giardia cysts in environmental samples, especially water. The inclusion of the vital dye propidium iodide (PI) by oocysts and cysts has been previously shown to correlate well with nonviability. The ability of nonviable oocysts and cysts to include PI has been employed to develop a rapid viability determination method that could be used in conjunction with the current indirect fluorescent antibody (IFA) method for detecting oocysts and cysts. The efficacy of this PI–IFA method to detect and determine the viability status of oocysts/cysts has been tested using oocyst samples inactivated by three different approaches. The ability to incorporate PI staining with IFA detection provides the advantage of both detection and viability determination at the same time, using the same sample.Key words: propidium iodide, viability, oocysts, cysts, detection.
The expression of nonagglutinating fimbriae and its role inProteus mirabilisadherence to epithelial cells
Authors: Tolson Douglas L. , Harrison Blair A. , Latta Roger K. , Lee Kok K. , Altman Eleonora
Published: February 2010

Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis.Key words: fimbriae, adherence, monoclonal antibodies, Proteus mirabilis, receptors.
Mutants in theSaccharomyces cerevisiae RAS2gene influence life span, cytoskeleton, and regulation of mitosis
Authors: Pichová Alena , Vondráková Dagmar , Breitenbach Michael
Published: February 2010

We investigated the phenotypic consequences in Saccharomyces cerevisiae of a disruption allele (ras2::LEU2) and of a dominant mutant form (RAS2ala18,val19) of RAS2. In addition to the phenotypes described earlier for these mutants, we observed a small increase in the life span for the disruption allele and a drastic decrease of life span for the dominant mutant form, as compared with the isogenic wild type. This was found by analyzing these alleles in two different genetic backgrounds with nearly the same results. Life spans were determined by micromanipulating mother cells and counting generations until no further cell division occurred. A morphological analysis of the terminal phenotypes of very old mother cells was performed showing enlarged or rounded cells and in some cases elongated buds, some of which were difficult to separate from the mother cell. This was observed in wild-type cells, as well as mutant cells. However, the dominant RAS2 mutant (but not the wild-type or ras2::LEU2 mutant cells) after 2 days on complex media displayed phenotypes similar to the terminal phenotype of old mothers. A substantial fraction of the cells were enlarged and generated elongated buds, they lost Calcofluor staining of the bud scars, the cell surface appeared folded, the actin cytoskeleton was aberrant, and the mitotic spindle and the cytoplasmic microtubules were defective in their proper orientation, resulting in aberrant mitoses and empty buds. These phenotypic characteristics of the RAS2ala18,val19mutation could be causative for the previously observed rapid loss of viability of these cells in stationary phase.Key words: yeast, Saccharomyces cerevisiae, RAS, oncogene, aging, morphology.
Isolation of endophytes from two species of palm, from Bermuda
Authors: Southcott K. A. , Johnson J. A.
Published: February 2010

This is the first report of endophytes from the indigenous Bermudian palmetto (Sabal bermudana Bailey) and the introduced Chinese palmetto (Livistona chinensis Jacquin), from Bermuda. Fronds were surface sterilized and 8-mm-diameter disks were removed and placed on 2% malt extract agar. Fungal isolates were obtained from 76 of the 375 disks from both species of palm. Idriella (two species) was the most common taxon isolated from both species of palm, making up 22 of the total 76 isolates, while Aspergillus accounted for 17 of the total 76 isolates. An unidentified isolate (BCP95-A), found in the Bermudian and the Chinese palmettos, accounted for 8 of the total 76 isolates. No statistically significant difference was found between fungal isolation frequencies of the two species of palm.Key words: endophyte, palm, leaves, Bermuda.
A gnotobiotic bioassay for studying interactions between potatoes and plant growth-promoting rhizobacteria
Authors: Conn Kenneth L. , Lazarovits George , Nowak Jerzy
Published: February 2010

A gnotobiotic bioassay, using potato plantlets derived from single-node explants and grown in test tubes containing potato nodal cutting medium (PNCM), was found to be highly useful for investigations of direct growth promotion by a nonfluorescent Pseudomonas sp. strain PsJN. Strain PsJN was used to optimize and evaluate this bioassay for purposes of screening other rhizosphere bacteria and identification of Tn5 mutants of strain PsJN deficient in growth-promoting properties. The selection of potato cultivar used in this bioassay was critical, as growth promotion of potatoes by strain PsJN was cultivar specific. Inoculated plantlets of cultivars Norchip, Kennebec, Shepody, and Chaleur showed, in root dry weight, a five- to eight-fold increase, two- to three-fold increase, no response, and a decrease of 50%, respectively. Haulm dry weight followed similar trends but was not as consistent an indicator of growth promotion. Bioassay results were not altered to any extent by minor changes in PNCM composition or by slight changes in temperature and light conditions. A rapid method for preparation of bacterial suspensions and inoculation of explants was developed. Inoculation of three explants taken from 6-week-old stock plantlets of cv. Kennebec for each Tn5 transconjugate of strain PsJN (total of 1500 transconjugates) enabled the elimination of 93% of those isolates that retained growth-promoting activity. The remaining 7% of isolates were retested and seven were confirmed to have lost growth-promoting ability. Bacteria from different genera were also screened with this bioassay. None of these bacteria increased the growth of potato plantlets, but several inhibited root and haulm growth.Key words: plant growth-promoting rhizobacteria, gnotobiotic, tissue culture, nonfluorescent pseudomonad, bacterium, potato.
Fate of three genetically engineered, biotechnologically important microorganism species in soil: impact of soil properties and intraspecies competition with nonengineered strains
Authors: Vahjen Wilfried , Munch Jean Charles , Tebbe Christoph C.
Published: February 2010

The fate of a bacterium and two yeast species genetically engineered by insertion of a nucleotide sequence encoding for aprotinin was studied in three different soils. Corynebacterium glutamicum carried the recombinant gene on plasmid pUN1, Saccharomyces cerevisiae carried the gene on plasmid p707, and in Pichia angusta (formerly Hansenula polymoropha) LR9-Apr8, the gene was chromosomally inserted with eight tandem repeats. Corynebacterium glutamicum persisted longer than both yeast strains. In a sandy loam of pH 5.9, recovery rates of cultured cells were lower than in a clay silt or a silty sand, with pH values of 7.1 and 6.7, respectively. Generally, persistence at 10 °C was higher than at 20 °C. An adaptation of the genetically engineered strains resulting in higher soil persistence was not observed for any of the three species tested. Competition experiments between nonengineered and genetically engineered strains in presterilized soils revealed a reduced fitness of the engineered strains. However, a more competitive C. glutamicum pUN1 evolved after reinoculation of cells, preselected by a preceding competition experiment.Key words: ecological risk assessment, genetic engineering, nondeliberate release, soil inoculation, aprotinin.