bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of diclofop-methyl by pure cultures of bacteria isolated from Manitoban soils
Authors: Smith-Grenier L. L. , Adkins A.
Published: February 2010

Pure cultures of Chryseomonas luteola and Sphingomonas paucimobilis isolated from Manitoban soils were able to utilize diclofop-methyl (methyl-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate) as the sole source of carbon and energy. An actively growing culture of C. luteola completely degraded 1.5 μg diclofop-methyl∙mL−1to diclofop acid and 4-(2,4-dichlorophenoxy)phenol within 71 h, as determined by gas chromatographic analysis. The accumulation of these metabolites in the growth medium resulted in the cessation of growth, indicating the organism's inability to degrade phenoxyphenol in the presence of diclofop acid. Sphingomonas paucimobilis mineralized 1.5 μg diclofop-methyl∙mL−1to diclofop acid within 54 h. A biphasic growth pattern indicated that this organism was capable of degrading diclofop acid to 4-(2,4-dichlorophenoxy)phenol and 2,4-dichlorophenol and (or) phenol. Neither of the organisms was able to utilize 2,4 -dichlorophenol as the sole source of carbon and energy.Key words: diclofop-methyl, biodegradation, herbicide.
Characterization of sulfate-reducing bacteria isolated from oil-field waters
Authors: Tardy-Jacquenod C. , Caumette P. , Matheron R. , Lanau C. , Arnauld O. , Magot M.
Published: February 2010

The occurrence and metabolic capacities of sulfate-reducing bacteria (SRB) were studied in 23 water samples taken from producing wells at 14 different sites. Oil fields in France, the North Sea, and the Gulf of Guinea were selected and classified according to physicochemical parameters (salinity ranging from 0.3 to 120 g∙L−1 and temperature between 29 and 85 °C). After the distribution of SRB within oil fields was studied, several strains of SRB were isolated and characterized metabolically. Twenty of the thirty-seven strains were not related to any known species. Most of the identified strains were members of the genera Desulfovibrio and Desulfotomaculum by molecular, morphological, and physiological properties.Key words: sulfate-reducing bacteria, oil-field ecology, metabolic identification, biodiversity.
Rhizobium leguminosarum as a plant growth-promoting rhizobacterium: direct growth promotion of canola and lettuce
Authors: Noel T. C. , Sheng C. , Yost C. K. , Pharis R. P. , Hynes M. F.
Published: February 2010

Early seedling root growth of the nonlegumes canola (Brassica campestris cv. Tobin, Brassica napus cv. Westar) and lettuce (Lactuca saliva cv. Grand Rapids) was significantly promoted by inoculation of seeds with certain strains of Rhizobium leguminosarum, including nitrogen- and nonnitrogen-fixing derivatives under gnotobiotic conditions. The growfh-promotive effect appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. Auxotrophic Rhizobium mutants requiring tryptophan or adenosine (precursors for indole-3-acetic acid and cytokinin synthesis, respectively) did not promote growth to the extent of the parent strain. The findings of this study demonstrate a new facet of the Rhizobium–plant relationship and that Rhizobium leguminosarum can be considered a plant growth-promoting rhizobacterium (PGPR).Key words: Rhizobium, plant growth-promoting rhizobacteria, PGPR, indole-3-acetic acid, cytokinin, roots, auxotrophic mutants.
An overview of microbial research related to high-level nuclear waste disposal with emphasis on the Canadian concept for the disposal of nuclear fuel waste
Authors: Stroes-Gascoyne Simcha , West Julia M.
Published: February 2010

Current research on the effects of microbiology on nuclear waste disposal, carried out in a number of countries, is summarized. Atomic Energy of Canada Limited has developed a concept for the permanent disposal of nuclear fuel waste in Canada. A program was initiated in 1991 to address and quantify the potential effects of microbial action on the integrity of the multibarrier system on which the disposal concept is based. This microbial program focuses on answering specific questions in areas such as the survival of bacteria under relevant radiation and desiccation conditions; growth and mobility of microbes in compacted clay buffer materials and the potential consequences for container corrosion and microbial gas production; the presence and activity of microbes in deep granitic groundwaters; and the effects of biofilms on radionuclide migration in the geosphere.Key words: nuclear waste disposal, radiation and desiccation effects, microbially influenced corrosion, radionuclide migration, gas production.
Monitoring deep subsurface microbiota for assessment of safe long-term nuclear waste disposal
Authors: White David C. , Ringelberg David B.
Published: February 2010

Microbes with their resistance to heat and radioactivity, if present and metabolically active, could have major effects on the safety of nuclear waste disposal by posing potential problems in long-term containment. This paper reviews the applicability of the signature lipid biomarker (SLB) analysis in the quantitative assessment of the viable biomass, community composition, and nutritional/physiological status of the subsurface microbiota as it exists in situ in subsurface samples. The samples described in this review are not unlike those expected to be recovered from proposed deep subsurface disposal sites. Assessment of the microbial community ecology using SLB analysis can be utilized to predict potential problems engendered by microbial metabolic activities of these communities in breaching containment by microbially facilitated corrosion and in the potential for subsequent facilitated transport of nuclides into the environment. SLB analysis of the in situ microbial ecology can be utilized to monitor the feasibility of containment options in modeling tests at the specific disposal sites.Key words: nuclear waste, deep subsurface, microbiota, microbial corrosion, safe long-term storage, signature lipid biomarkers.
The geochemical environment of nuclear fuel waste disposal
Authors: Gascoyne M.
Published: February 2010

The concept for disposal of Canada's nuclear fuel waste in a geologic environment on the Canadian Shield has recently been presented by Atomic Energy of Canada Limited (AECL) to governments, scientists, and the public, for review. An important part of this concept concerns the geochemical environment of a disposal vault and includes consideration of rock and groundwater compositions, geochemical interactions between rocks, groundwaters, and emplaced vault materials, and the influences and significance of anthropogenic and microbiological effects following closure of the vault. This paper summarizes the disposal concept and examines aspects of the geochemical environment. The presence of saline groundwaters and reducing conditions at proposed vault depths (500–1000 m) in the Canadian Shield has an important bearing on the stability of the used nuclear fuel, its container, and buffer and backfill materials. The potential for introduction of anthropogenic contaminants and microbes during site investigations and vault excavation, operation, and sealing is described with examples from AECL's research areas on the Shield and in their underground research laboratory in southeastern Manitoba. Keywords: nuclear waste disposal, geochemistry, Canadian Shield, groundwater chemistry.
Purification and characterization of an extracellular phenol oxidase from culture filtrates ofPyricularia oryzae
Authors: Alsubaey Aisha , Sisler Hugh D. , Matthews Benjamin F.
Published: February 2010

Extracellular phenol oxidase activity was characterized and compared in Pyricularia oryzae wild-type and albino cell types to determine if this phenol oxidase was responsible for lack of melanization in the albino culture. Filtrates of the albino mutant Alb-5 showed activity similar to those of the wild type, while those of a buff mutant (Cp62) showed weak phenol oxidase activity. This indicated that the lack of melanization in the albino mutant was not due to an absence of phenol oxidase activity. The phenol oxidase isoform patterns from the wild type and two mutants were similar when analyzed by polyacrylamide gel electrophoresis. The slowest migrating isoform of phenol oxidase from wild-type Pyricularia oryzae was the major form and had a molecular mass of 380 kDa. The molecular masses of two of the minor forms were 220 and 130 kDa. The isoforms oxidized 1,8-dihydroxynaphthalene, the terminal metabolite in the polyketide pathway to melanin. The major phenol oxidase isoform was also present in extracts from albino mutants and the buff mutant. The major form was enriched by a combination of ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and elution from preparative polyacrylamide gels. The enriched isoform of phenol oxidase separated into two forms after a second electrophoresis, indicating that these two isoforms interconvert. Analysis of both forms by sodium dodecyl sulfate – polacrylamide gel electrophoresis indicated that both were composed of a single subunit with a molecular mass of 70 kDa. The enriched isoform preferred phloroglucinol as a substrate and had a Michaelis constant (Km) of 19.3 mM for phloroglucinol and a pH optimum between 6 and 7.5.Key words: phenol oxidase, laccase, Pyricularia oryzae, rice blast, melanin.
Negative chemotaxis inCytophaga johnsonae
Authors: Liu Zheng-Xian , Fridovich Irwin
Published: February 2010

Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria. The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl−, and N-chlorotaurine, as well as by low pH. Its response to H2O2was eliminated by catalase. Nalidixic acid at 200 μM, which inhibits the growth but not the motility of C. johnsonae, did not interfere with its negative chemotactic response to H2O2, whereas sodium phosphate at 10 mM, which inhibits motility, did so. Cytophaga johnsonae was not repelled by taurine, n-octanol, phenol, L-valine, or high pH. Chemotaxis can be conveniently studied in gliding bacteria such as C. johnsonae.Key words: gliding bacteria, Cytophaga johnsonae, negative chemotaxis, hydrogen peroxide, N-chlorotaurine.
Influence of nutrients on growth ofEpicoccum nigrum
Authors: Zhou Ting , Reeleder R. D. , Sparace S. A.
Published: February 2010

Epicoccum nigrum is a potential biological control agent for certain plant diseases, such as white mold of bean caused by Sclerotinia sclerotiorum. To provide information that could be used to improve the production and efficacy of E. nigrum, the effects of nutrients, including specific carbohydrate sources and amino acids, on mycelial growth, sporulation, germination of conidia, and elongation of germ tubes were determined. In dual cultures of E. nigrum and S. sclerotiorum, the effects of nutrients on widths of inhibition zones between the two fungi were assessed. Standard mycological media supported faster radial growth than media with single carbohydrate sources and individual amino acids, but glutamic acid combined with maltose or dextrose was similar with respect to stimulation of sporulation when compared with media such as V8 juice and yeast extract agars. Dual culture inhibition zones were greater in certain simple media (dextrose and lysine, sucrose and lysine, and maltose and lysine) than in standard media. For germination and germ tube elongation, sucrose and maltose were superior to most other carbohydrate sources tested, and lysine and glutamic acid were superior amino acid sources. When standard broth media were compared for production of antifungal compounds by E. nigrum, both potato dextrose broth and malt extract broth were superior to Czapek solution. Culture filtrates of E. nigrum grown in potato dextrose broth were more inhibitory towards S. sclerotiorum than filtrates from malt extract cultures.Key words: biological control, white mold, Epicoccum purpurascens, antibiosis.
An improved microinjection protocol for the transfer ofWolbachia pipientisbetween infected and uninfected strains of the flour beetleTribolium confusum
Authors: Chang Nancy W. , Wade Michael J.
Published: February 2010

The lethality of halocarbon and other oils to hatching larvae of the flour beetle Tribolium confusum limits existing microinjection protocols, because postinjection survivorship is only 5.6% of the eggs injected. We report the development of an oil-free protocol that improves survivorship fivefold. We used this protocol to transfert the cytoplasmic endosymbiont Wolbachia pipientis from infected donor eggs to uninfected host eggs and observed reproductive incompatibility in 40% of the surviving, injected eggs. Compared with mock injected controls (35.9% survival) or microinjection of uninfected cytoplasm (32.3% survival), injection of Wolbachia-infected cytoplasm into host eggs significantly reduced egg-to-adult survival (25.1%).Key words: microinjection, Wolbachia, transfection, flour beetle, Tribolium confusum.
Nested PCR protocol for the rapid detection of Escherichia coli in potable water
Authors: Juck David , Ingram Jordan , Prévost Michèle , Coallier Josée , Greer Charles
Published: February 2010

A rapid and sensitive method for the detection of low levels of bacteria in potable water was developed. The fecal indicator bacterium Escherichia coli was used as the test organism in a filtration concentration–nested polymerase chain reaction (PCR) protocol, combined with ethidium bromide visualization of PCR products. Two sets of primers were designed from the E. coli specific β-glucuronidase gene (uidA), the primary pair producing a 486-bp fragment that was used as template for the nested primer pair delineating a 186-bp fragment. This protocol can detect 1–10 bacterial cells/50 mL water sample within 6–8 h, in contrast to traditional culturing or Southern hybridization methods which require 2–3 days for results.Key words: nested PCR, sensitive, detection, potable water.
Oxidation and amidation of salicylate by Streptomyces species
Authors: Pospíšil Stanislav , Přikrylová Věra , Němeček Jan , Spížek Jaroslav
Published: February 2010

Seven streptomycete strains were tested for biotransformation of salicylate. The products were identified by nuclear magnetic resonance spectroscopy and three types of conversion were found. Streptomyces cinnamonensis and Streptomyces spectabilis formed gentisate and salicylamide concurrently. Streptomyces rimosus transformed salicylate to salicylamide. Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus and Streptomyces avermitilis produced only gentisate. Time course studies of salicylate conversion by thin-layer chromatography and high pressure liquid chromatography showed that salicylamide was accumulated in the culture broth, whereas gentisate was further metabolized.Key words: salicylate, gentisate, salicylamide, biotransformation, Streptomyces spp.
Germination of soil-incorporated microsclerotia of Colletotrichum truncatum and colonization of seedlings of the weed Sesbania exaltata
Authors: Schisler David A. , Jackson Mark A.
Published: February 2010

Microsclerotia of the bioherbicidal fungus Colletotrichum truncatum were produced in submerged culture. Seedlings of the weed Sesbania exaltata became infected when seeds were germinated in air-steam pasteurized (60 °C, 30 min) field soil infested with 165 microsclerotia/cm3. Infection was first noted 3 days after planting seeds, when the pathogen was recovered from 5% of plant segments taken from within 0.5 cm of the soil surface. By day 7, C. truncatum was recovered from 38% of stem and root segments within 0.5 cm of the soil surface, and from 60% of similar segments by day 8. Of all pathogen recovery, 66% came from segments within 0.5 cm of the soil surface and 92% of recoveries came from within 1.0 cm of the soil surface. All freshly produced microsclerotia on Nobel water agar had germinated after 24 h and conidial production from germinated microsclerotia was detected. Conidiation peaked in vitro after 2 days, with approximately 3500 conidia being produced per microsclerotium. In situ, light microscopy showed that about 40% of microsclerotia produce setae after 1 day in pasteurized potting mix. This level was virtually unchanged after 2, 3, and 4 days of incubation though all microsclerotia remained viable. Scanning electron microscopy determined that, in situ, microsclerotia germinated sporogenically to produce conidia and setae. Newly produced conidia germinated and formed appressoria on S. exaltata roots after 2 days, when root radicles were less than 1 day old.Key words: Colletotrichum truncatum, Sesbania exaltata, bioherbicide, mycoherbicide, microsclerotia.
Purification and characterization of an endo-β-1,3-glucanase fromTrichoderma harzianum
Authors: Noronha Eliane Ferreira , Ulhoa Cirano José
Published: February 2010

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Kmand Vmaxvalues for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2and stimulated by cations such as Zn2+and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.
In vitro degradation of dicyclopentadiene by microbial consortia isolated from hydrocarbon-contaminated soil
Authors: Stehmeier L. G. , Jack T. R. , Voordouw G.
Published: February 2010

Degradation of dicyclopentadiene (DCPD) to carbon dioxide and oxygenated intermediates was established in the laboratory. Screening of many inocula using BIOLOG™ MT plates showed that no single colony isolate readily mineralized DCPD. Mixed cultures from a variety of environmental sources produced 14CO2 when incubated with [14C]DCPD, but most of the DCPD was metabolized to oxygenated intermediates that could be extracted from the culture liquid and detected using gas chromatography and mass spectroscopy. Stimulation of environmental inocula with nutrients and preexposure to DCPD before testing for degradation gave mineralization rates after 25 days of in vitro incubation that were twice as fast as those previously reported.Key words: dicyclopentadiene, bioremediation, microbial community, GC–MS, biometer flask.
Expression of T-cell receptor Vβ2 and type 1 helper T-cell-related cytokine mRNA in streptococcal pyrogenic exotoxin-C-activated human peripheral blood mononuclear cells
Authors: Ohara-Nemoto Y. , Kaneko M.
Published: February 2010

Streptococcal pyrogenic exotoxin type C (SPE C) is a member of the bacterial superantigens that are potent stimulants of T cells. We expressed SPE C in Escherichia coli and characterized its selective stimulation properties on human T cells bearing specific Vβ chains of T-cell receptors (TCRs). Cytokine profiles induced by SPE C were also examined. Recombinant SPE C significantly enhanced proliferation of human peripheral blood mononuclear cells (PBMCs) at concentrations as low as 10−12–10−14 M. Reverse transcription of RNA from SPE-C-stimulated PBMCs followed by polymerase chain reaction, revealed selective induction of TCR Vβ2 chain expression. SPE C raised the mRNA level of type 1 helper T cell (TH1) related cytokines, such as interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor β (TNFβ). The expression of TNFα was also increased. In contrast, the increase in mRNA levels of the p35 small fragment of IL-12 and type 2 helper T cell (TH2) related cytokines (i.e., IL-4 and IL-10) was not significantly affected by SPE C. The mRNA level of proinflammatory cytokine IL-6 was increased marginally. Consistent with the mRNA accumulation, protein concentrations of IFNγ, IL-2, and TNF were increased in SPE-C-stimulated PBMCs, but IL-4 was not. From these results, we conclude that the stimuli of SPE C preferentially causes the TH1 responses in human T cells bearing TCR Vβ2.Key words: streptococcal pyrogenic exotoxin C, Streptococcus pyogenes, T cells, cytokine, superantigen.
Biosynthesis of amino acids byOxalobacter formigenes: analysis using13C-NMR
Authors: Cornick Nancy A. , Yan Bin , Bank Shelton , Allison Milton J.
Published: February 2010

The gram-negative anaerobe Oxalobacter formigenes, grows on oxalate as the principal carbon and energy source, but a small amount of acetate is also required for growth. Experiments were conducted to determine the distribution and the position of label in cellular amino acids from cells grown on [13C]oxalate, [13C]acetate (1-13C, 2-13C, and U-13C), and13CCO3. The labeling pattern (determined with NMR spectroscopy) of amino acids was consistent with their formation through common biosynthetic pathways. The majority of the carbons in the amino acids that are usually derived from pyruvate, oxaloacetate, α-ketoglutarate, 3-phosphoglycerate, and carbon in the aromatic amino acids were labeled by oxalate. Carbon from13CO3was assimilated primarily into amino acids expected to be derived from oxaloacetate and α-ketoglutarate. Approximately 60% of the acetate that was assimilated into amino acids was incorporated as a C2unit into proline, arginine, glutamate, and leucine. The pattern of labeling from acetate in glutamate, arginine, and proline was consistent with acetate incorporation via citrate (si)-synthase and subsequent formation of α-ketoglutarate via the first third of the tricarboxylic acid pathway. Acetate was also assimilated into amino acids derived from pyruvate and oxaloacetate, but results indicated that this incorporation was as single carbon atoms. Based on these findings, cell-free extracts were assayed for several key biosynthetic enzymes. Enzymatic activities found included glutamate dehydrogenase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase. These findings are consistent with proposed biosynthetic mechanisms.Key words: oxalate, carbon flow, carbon assimilation.
Nodulation ofPhaseolus vulgarisbyRhizobium etliis enhanced by the presence ofBacillus
Authors: Srinivasan M. , Holl F. B. , Petersen D. J.
Published: February 2010

The ability of Bacillus spp. to alter the nodulation of Phaseolus vulgaris by Rhizobium etli was assessed. The simultaneous presence of both Rhizobium etli TAL 182 and Bacillus megaterium S49 on plant roots during the early stages of plant growth was necessary for enhanced nodulation of Phaseolus vulgaris by the Rhizobium microsymbiont. Coinoculation with both bacterial species also facilitated heterologous nodulation of Rhizobium TAL 182 on Phaseolus acutifolius. These results are consistent with earlier reports of increased root hair proliferation and lateral root formation in response to coinoculation. Split-root experiments revealed that coinoculation partially suppressed host-controlled regulation of nodulation, implicating a plant interaction with the two bacterial species. Changes to the nodulation potential of R. etli due to coinoculation with Bacillus spp. demonstrate the potential for root-associated organisms other than rhizobia to alter the dynamics of the legume–Rhizobium symbiosis.Key words: Bacillus, nodulation enhancement, heterologous nodulation.
The 24-kDa protein from Fusarium oxysporum f.sp. erythroxyli: occurrence in related fungi and the effect of growth medium on its production
Authors: Bailey Bryan A. , Jennings James C. , Anderson James D.
Published: February 2010

A 24-kDa protein that elicits ethylene production and necrosis in leaves of dicotyledonous plants was previously purified from culture filtrates of Fusarium oxysporum Schlechtend:Fr. f.sp. erythroxyli. Antisera to the denatured 24-kDa protein detected 2.5 ng of the 24-kDa protein on Western blots at 100 000-fold dilutions. The antisera cross-reacted with a 24-kDa protein on Western blots of culture filtrates from three other F. oxysporum formae speciales. Of seven Fusarium species, only F. oxysporum, F. acuminatum Ellis and Kellerm., and F. avenaceum (Fr.:Fr.) Sacc. isolates produced an antigenically related 24-kDa protein. Although there were differences in the profiles of proteins extracted from stems of coca (Erythroxylum coca var. coca L. Lam.) infected with F. oxysporum f.sp. erythroxyli compared with uninfected stems, antisera to the 24-kDa protein did not cross-react with any proteins from the infected coca stems. For the fungal isolates studied, the best medium tested for production of the 24-kDa protein contained 1% sucrose and 1% asparagine. Biological activity of the F. oxysporum culture filtrates on sweet basil leaves was consistently correlated with the presence of the 24-kDa protein. Production of the 24-kDa protein was limited in cultures containing pectin or cellulose as the primary carbon source, or in cultures lacking sucrose or casamino acids. Water-soluble extracts from coca stems inhibited production of the 24-kDa protein, whereas cellulose and pectin did not. Components produced by the plant may limit production of the 24-kDa protein in infected plant tissue and thereby limit the response of the plant to the fungus. These results suggest the 24-kDa protein does not function in the symptomatic phase of the F. oxysporum f.sp. erythroxyli–coca disease interaction.Key words: Fusarium oxysporum, toxin, elicitor.
A rapid and efficient system for screening HIV-1 Pol mRNA-specific ribozymes
Authors: Ramezani A. , Marhin W. , Weerasinghe M. , Joshi S.
Published: February 2010

Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs. However, the reports that exist on their activity against different targets have described mixed success. As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzProdirected against the protease (Pro) coding region and RzRTdirected against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli. Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro. RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzProand RzRTtarget sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT. The RT activity in cells expressing RzRTwas shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any, that could enhance ribozyme activity in vivo.Key words: HIV, hammerhead ribozyme, protease, reverse transcriptase.
Immunolocalization of fimbrial epitopes in thin sections of Microbotryum violaceum
Authors: Celerin Martina , Day Alan W. , Smith Ronald J. , Laudenbach David E.
Published: February 2010

Fungal fimbriae are long (0.5–20 μm), narrow (7 nm) surface appendages that have been observed on most members of the Mycota. Biochemical analyses have determined that fimbriae from Microbotryum violaceum are composed of 74-kDa glycoproteinaceous subunits in which the protein moiety is fungal collagen. We present evidence for the localization of fimbrial subunits prior to their exportation from the cell. We term these internal, likely nonpolymerized fimbriae "pro-fimbriae" and demonstrate the location of the reserves within the peripheral cytoplasm. Also, we show that fimbriae may not traverse the cell wall as previously believed, but may instead originate from within the outer lamella of the cell wall, possibly being anchored to the cell wall via other molecules. This model is analogous to the animal extracellular matrix arrangement in which collagens are anchored to plasma membranes via other proteins such as fibronectin.Key words: fungus, immunolocalization, fimbriae, Microbotryum, Ustilago.
Bacterial endophytes in cotton: location and interaction with other plant-associated bacteria
Authors: Quadt-Hallmann A. , Hallmann J. , Kloepper J. W.
Published: February 2010

Investigations were conducted to determine if biological control agent Pseudomonas fluorescens 89B-61 could colonize cotton tissues systemically and if internal colonization by a known endophytic bacterium, Enterobacter asburiae JM22, was influenced by the presence of other plant-associated bacteria. Following seed treatment, Pseudomonas fluorescens 89B-61 colonized cotton roots both externally and internally at mean population densities of 8.7 × 105 CFU/g and 1.1 × 103 CFU/g, respectively. However, bacteria were not detected in cotyledons, leaves, or stems. After inoculation onto leaves, Pseudomonas fluorescens 89B-61 established a mean internal population density of 1.6 × 104 CFU/g leaf tissue. Following stem injection, Pseudomonas fluorescens 89B-61 did not colonize roots or leaves. Pseudomonas fluorescens 89B-61 was localized on the root surface concentrated in grooves between epidermal cells, below collapsed epidermal cells, and in intercellular spaces close to the root epidermis, as identified by immunogold labeling of the bacterial membrane. Combined application of E. asburiae JM22 with another endophyte, Paenibacillus macerans Tri2-10, resulted in significantly lower internal populations of E. asburiae JM22 compared with treatment with E. asburiae JM22 alone. However, when coinoculated with a rhizosphere colonist, Micrococcus agilis strain 2RD-11, the colonization density of E. asburiae JM22 was not negatively affected. The results suggest that the internal colonization of cotton by bacteria with biological control activity may be an important aspect in their capacity to protect host plants against plant pathogens. The extent of internal colonization was shown to be influenced by other bacterial colonists.Key words: endophytic bacteria, location, interaction, cotton.
Production ofD-amino acid oxidase byAspergillussp. strain O20 active on cephalosporin C
Authors: Mujawar Salim K. , Shewale Jaiprakash G.
Published: February 2010

Aspergillus sp. strain O20 produces inducible D-amino acid oxidase intracellularly, only in the presence of some amino acids. The enzyme was induced most effectively by the addition of DL-alanine (1% w/v) to the production medium. Among the various compounds studied, production of the D-amino acid oxidase was enhanced by Aerosol-22, glucose, and sodium nitrate. D-Amino acid oxidase formation was observed during the onset of the stationary growth phase. Maximum enzyme activity was recorded after 96 h of fermentation (1000 IU/L).Key words: D-amino acid oxidase, Aspergillus sp., 7-aminocephalosporanic acid, cephalosporin C.
Cloning and sequencing of the hyaluronate lyase gene fromPropionibacterium acnes
Authors: Steiner Bret , Romero-Steiner Sandra , Cruce Donna , George Robert
Published: February 2010

The hyaluronate lyase (hyaluronidase) gene from Propionibacterium acnes was cloned and sequenced. The gene was isolated on an EcoRI-generated 3-kb piece of DNA. Expression was less in Escherichia coli than in P. acnes; in E. coli, active enzyme was only cell associated and not secreted. The gene is 2256-bp long and codes for a protein of 82 kDa. Amino terminal sequencing of the protein of the partially purified gene indicated the presence of a 32-amino-acid leader sequence. The leader sequence showed a membrane-spanning domain and all of the features usually associated with the leader for a secreted protein. The amino acid sequence is predicted to share homology with the hyaluronidase enzymes from Streptococcus pneumoniae, Streptococcus agalactiae, and Staphylococcus aureus. A potential hyaluronate-binding domain was identified and antibody against this domain was inhibitory to the enzyme.Key words: Propionibacterium acnes, hyaluronidase, leader sequence, hyaluronate binding domain, sequence homology among hyaluronidases.
Recherche de souches naturelles du Bacillus thuringiensis dans différents biotopes, à travers le monde
Authors: Chaufaux Josette , Marchal Michel , Gilois Nathalie , Jehanno Isabelle , Buisson Christophe
Published: February 2010

A screening programme to isolate new strains of the entomopathogenic bacteria Bacillus thuringiensis was undertaken on 4887 samples of various sources from 101 countries over the world: 1260 strains of the bacillus were isolated. Dust from mills and silos, as well as insects from nature, were more successful sources than soil samples, which emphasizes the diversity of biotopes where the bacillus is encountered. Electrophoretic characterization reveals the genetic variability of the species. An analysis of insecticidal properties of the isolated strains was performed on four insect species: Plutella xylostella (Lepidoptera: Plutellidae), Spodoptera littoralis (Lepidoptera: Noctuidae), Phaedon cochleariae (Coleoptera: Chrysomelidae), and Locusta migratoria or Schistocerca gregaria (Orthoptera: Acrididae). The most frequent strains (54%) were producing crystals constituted of proteins with molecular masses of 130–140 or 66 and 130–140 kDa and were toxic to Lepidoptera larvae. A significant number of strains (31) were larvicidal to Coleoptera while only one, H14 serotype, was active on Diptera. Numerous strains synthesize crystals made up of proteins with size differing from the already known toxins. Most of these strains were nonactive against the four insect species tested. One strain showing a protein band at 73 kDa had no insecticidal activity against P. cochleariae while it was toxic to P. xylostella.Key words: Bacillus thuringiensis, new strains, distribution, Lepidoptera, Coleoptera.
Characterization ofBacillus thuringiensismutants and natural isolates by molecular methods
Authors: Jung Yong Chul , Kim Sung Uk , Bok Song Hae , Park Ho Yong , Côté Jean-Charles , Chung Young Sup
Published: February 2010

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryI- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryI and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Another kurstaki HD-1 mutant, called NGK-13 and obtained following treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous–acrystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-type genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA(c) genes. Isolate BT-14 contained only the cryIA(c) gene, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.Key words: Bacillus thuringiensis, δ-endotoxin crystal, cry-type genes, polymerase chain reaction.
Environmental mycobacteria in bottled table waters in Greece
Authors: Papapetropoulou M. , Tsintzou A. , Vantarakis A.
Published: February 2010

A hundred and fifty samples of bottled table water sold by Greek factories were examined for the presence of environmental mycobacteria. Environmental mycobacteria were found in 23 (15.6%) of the 150 tested samples. Bacterial numbers of 1–100, 101–300, 301–1000, and > 103CFU/L were found in 8, 2, 1, and 4% of the samples, respectively. The identification of the environmental mycobacteria was performed by both polymerase chain reaction – restriction enzyme analysis (PCR–REA) and biochemical methods. The environmental mycobacteria found were 14 Mycobacterium chelonae, 3 Mycobacterium phlei, 4 Mycobacterium gordonae, and 2 Mycobacterium flavescens. The relatively high number of environmental mycobacteria in bottled table water leads us to believe that the search of these opportunistic microorganisms in bottled water could be a useful index of their hygienic quality when this water is to be consumed by immunologically compromised patients. No statistically significant correlation was found between the presence of mycobacteria and the bacteriological faecal indicators (P < 0.005).Key words: environmental mycobacteria, bottled water, PCR–REA, Greece.
The sap-staining fungus Ophiostoma piceae synthesizes different types of melanin in different growth media
Authors: Eagen Rebecca , Brisson Anne , Breuil Colette
Published: February 2010

Sap-staining fungi, in particular Ophiostoma piceae, cause significant economic problems for Canada's lumber industry. For O. piceae and a few other staining fungi, the dark pigment is melanin. The choice of carbon and nitrogen sources added as supplements to a basic liquid mineral medium affected the colour and characteristics of the pigment produced by O. piceae. The mycelia became coloured early during the active growth phase and the colour reached a maximum intensity during the stationary growth phase.Key words: melanin, growth, Ophiostoma piceae, pigmentation, sap-staining fungus.
Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface
Authors: Manocha M. S. , Xiong D. , Govindsamy V.
Published: February 2010

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.
Two small linear plasmids of Streptomyces jumonjinensis
Authors: Netolitzky Donald J. , Jensen Susan E. , Roy Kenneth L.
Published: February 2010

In a survey of plasmids in a variety of β-lactam antibiotic producing Streptomyces spp., two small linear plasmids (pSJL1 and pSJL2) of approximately 12 and 17.5 kb were detected within Streptomyces jumonjinensis NRRL 5741, in addition to the previously reported giant linear plasmids pSJL3 and pSJL4. Characterization of these plasmids by Southern hybridization indicated that no significant homology exists between the S. jumonjinensis plasmids and plasmids detected in other β-lactam antibiotic producing Streptomyces spp. Single and double restriction endonuclease digestions were performed to generate maps of the two plasmids. The plasmids pSJL1 and pSJL2 have copy numbers of 21–27 and 15–20, respectively.Key words: Streptomyces, linear plasmid, DNA hybridization, DNA homology.
Molecular characterization of coat protein genes of serologically distinct isolates of potato virus Y necrotic strain
Authors: Dhar A. K. , Singh R. P.
Published: February 2010

The coat protein (CP) genes of two potato virus Y necrotic isolates (N27 and a mutant strain N27-92), which differed in their reactivity to a monoclonal antibody (mab), were characterized. Both isolates could be detected by mab 4E7, but mab VN295.5 selectively reacted to N27 and not to N27-92. The CP genes of both isolates coded for 267 amino acids with ~99.0% identity at both the nucleotide and the amino acid levels. Nucleotide sequence comparison indicated five substitutions in N27-92 compared with N27. Three of these changes resulted in substitution of amino acids. Two transitions (A→G) in N27-92 changed threonine to alanine and lysine to arginine at positions 7 and 55, respectively, whereas a A→T transversion changed asparagine to isoleucine at position 27. The surface probability curves of both the isolates could almost be superimposed, except at amino acid positions 7 and 27. Since amino acid substitution at position 55 is conservative, changes from polar to hydrophobic amino acids (threonine→alanine and asparagine→isoleucine) at positions 7 and 27 might have changed the epitope(s) of N27-92, abolishing its detection by mab VN295.5.Key words: potato virus Y, PVYN, coat protein gene.
Cytophaga hutchinsoniiATCC 33406 contains a structural variant of the sulfonolipidN-acylcapnine
Authors: Drijber R. A. , McGill W. B.
Published: February 2010

Gliding bacteria of the genera Cytophaga and Flexibacter contain unusual sulfonolipids, called capnoids, in their outer membrane. The parent compound capnine has been identified as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Structural studies on N-acylaminosulfonates purified from a cellulolytic cytophaga, Cytophaga hutchinsonii ATCC 33406, revealed a structural variant of the aminosulfonate core in addition to a unique 2-hydroxy fatty acyl group, i17:0(2-OH), in amide linkage with the core. The source of the structural variation in the aminosulfonate core of Cytophaga hutchinsonii arose from the addition of two carbons to the acyl chain and the insertion of two trans-double bonds. Thus, the aminosulfonate core purified from Cytophaga hutchinsonii was identified as 2-amino-3-hydroxy-tras, trans-17-methylocta-4,7-diene-1-sulfonic acid and assigned the trivial name capnosine. A second cellulolytic cytophaga, CyP2, also showed this structural variation, which was absent from the noncellulolytic isolate CyS1. Further structural studies on sulfonolipids isolated from other cellulolytic and noncellulolytic isolates will determine the taxonomic significance of this structural variant of capnine.Key words: N-acylcapnine, N-acylaminosulfonates, capnosine, Cytophaga, cellulolytic cytophagas.
In situ enrichment and isolation of thermophillic microorganisms from deep-sea vent environments
Authors: Marteinsson Viggó Thór , Birrien Jean-Louis , Prieur Daniel
Published: February 2010

An in situ sampler that encloses individual blocks of thermally stable solid medium made with Gelrite was successfully deployed at a deep-sea hydrothermal vent environment. A mixed population of microorganisms was enriched on the surface of the Gelrite blocks. Thermophilic Bacilli spp. and bacteria belonging to the Thermotogales were isolated.Key words: in situ enrichment, sampler, hydrothermal vent.
Genetic and molecular analysis of Aspergillus nidulans transformants obtained by the biolistic process
Authors: Fungaro Maria Helena P. , Poli Regina C. , Pizzirani-Kleiner Aline A. , Azevedo João Lucio , Besin Evelyne , Lejeune Bernard
Published: February 2010

Twenty transformants obtained by transforming the argB strain of Aspergillus nidulans by the biolistic process were analysed in detail. Eight lost the Arg+ phenotype very frequently and the other 12 were mitotically stable, even after 15 successive subculturing. To analyse the integration events, the mitotically stable transformants were submited to genetic and molecular analyses; 16.7% resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus, 8.3% showed replacement of the argB mutation by wild type allele, 41.7% showed transforming DNA integrated into nonhomologous chromosomal regions at chromosome III, and 33.3% showed transforming DNA integrated into nonhomologous chromosomal regions at chromosomes others than III. Among the mitotically stable transformants, the frequency of integration into nonhomologous sites was higher by the biolistic process than that reported by the literature for protoplast-mediated transformation.Key words: Aspergillus nidulans, fungal transformation, biolistic gene transfer, gene integration, argB gene.
A conserved cysteine residue in yeast uroporphyrinogen decarboxylase is not essential for enzymatic activity
Authors: Flumeri Celestino Di , Acheson Nicholas H. , Keng Teresa
Published: February 2010

Uroporphyrinogen decarboxylase catalyzes the fifth step of heme biosynthesis in Saccharomyces cerevisiae. Studies utilizing sulfhydryl-specific reagents suggest that the enzyme requires a cysteine residue within the catalytic site This hypothesis was tested directly by site-directed mutagenesis of highly conserved cysteine-52 to serine or alanine. Plasmids containing these mutations were able to complement a hem6 mutant strain. In addition, properties associated with decreased uroporphyrinogen decarboxylase activity were not detected in the mutant strain transformed with these mutant plasmids. These results suggest that the conserved cysteine-52 by itself is not essential for enzymatic activity.Key words: heme biosynthesis, uroporphyrinogen decarboxylase, site-directed mutagenesis.