bluuebunny/crossref_metadata_2025 huggingface dataset:
Persistence in soil of transgenic plant producedBacillus thuringlensisvar.kurstakiδ-endotoxin
Authors: Palm C. J. , Seidler R. J. , Schaller D. L. , Donegan K. K.
Published: February 2010

Transgenic plants that produce pesticidal proteins will release these proteins into the soil when these plants are incorporated into the soil by tillage or as leaf litter. Little is known about the fate and persistence of transgenic plant pesticidal products in the soil. We used a model system of transgenic cotton that produces Bacillus thuringlensis var. kurstaki δ-endotoxin (Btk toxin) to evaluate the persistence of transgenic pesticides in soil. Purified Btk toxin or transgenic cotton leaves containing Btk toxin were added to soil in five different microcosm experiments in concentrations ranging from 1 to 1600 ng Btk toxin/g soil. The concentration of the extractable Btk toxin was measured for up to 140 days. An initial rapid decline in extractable toxin concentration in the first 14 days, followed by a slower decline, was observed in four of the five experiments. At the end of the experiments, Btk toxin from transgenic plant tissue was undetectable (less than 0.1% of starting concentration) in two of the microcosm experiments and at 3, 16, and 35% of the original amounts in the other experiments. In addition, experiments using γ-irradiated sterilized soil indicated that the observed decline in extractable toxin concentration was due largely to biotic degradation rather than to physical adsorption by the soil.Key words: transgenic plants, Bacillus thuringlensis toxin, risk assessment.
Development of engineered genomic DNA to monitor the natural transformation ofPseudomonas stutzeriin soil-like microcosms
Authors: Paget Eric , Simonet Pascal
Published: February 2010

The goal of this paper was to demonstrate whether natural transformation could occur in the environment to promote horizontal gene transfer between bacteria. Microcosms consisting of clay, clay and humic acids, or sterile soil were compared with respect to the natural transformation of Pseudomonas stutzeri by mineral-adsorbed DNA. Genes conferring resistance to tetracycline and ampicillin were first inserted in P. stutzeri pp100 chromosome via the pSUP202 suicide plasmid. Then, DNA extracted from the engineered P. stutzeri strain was used for transformation experiments, allowing the new transformed cells to be detected by hybridization with a tet probe. It turned out that DNA adsorbed on clay or soil particles and in presence of humic acids still transformed competent cells with frequencies up to 10−8transformants/viable cell. Finally, natural transformation assays involving two different DNAs were carried out in sterile soil microcosms. The use of nonisogenic DNA extracted from a rifampicin-resistant Pseudomonas fluorescens strain resulted in production of transformants, while isogenic DNA from our engineered strain failed to produce any. These observations confirmed that extracellular DNA adsorbed on a soil matrix composed of minerals and organic matter could still transform competent bacteria under environmental conditions.Key words: transformation, Pseudomonas stutzeri, soil microcosm, DNA, suicide plasmid.
Nitrogen source regulates expression of alanine dehydrogenase isoenzymes in Streptomyces avermitilis in a chemically defined medium
Authors: Novák Jan , Kopecký Jan , Vaněk Zdenko
Published: February 2010

Ammonium ions and alanine influence production of the macrolide avermectin in Streptomyces avermitilis. L-Alanine dehydrogenase and alanine aminotransferase are the primary enzymes responsible for regulating the intracellular concentration of alanine and also of ammonium ions. In cultures of S. avermitilis in a chemically defined medium with ammonia or L-alanine as the only nitrogen source, specific activities of both enzymes increased during growth. The alanine dehydrogenase specific activity increased more than 86-fold after the culture was supplemented with 0.2% L-alanine and 5-fold after addition of 0.5% ammonium sulfate, whereas alanine aminotransferase specific activity increased 3- to 4-fold with either substrate. Five isoenzymes of alanine dehydrogenase were detected histochemically in S. avermitilis after native gel electrophoresis. Isoenzyme 1 was induced by alanine and temporarily repressed by high concentrations of ammonium sulfate. The presence of isoenzyme 1 was also related to changes in the kinetic properties of the alanine dehydrogenase reaction measured in crude desalted extracts. A nonlinear double-reciprocal plot was obtained in initial velocity studies using L-alanine as a substrate in the sample induced with L-alanine. The nonlinearity was caused by both substrate inhibition and allosteric regulation (positive cooperativity) by L-alanine. In contrast, the sample induced by ammonium sulfate showed a linear double-reciprocal plot.Key words: isoenzymes, L-alanine dehydrogenase, Streptomyces avermitilis, avermectin.
Transformation of methylotrophic bacteria by electroporation
Authors: Gliesche Christian G.
Published: February 2010

An efficient system for electroporation of the methylotrophic bacteria Hyphomicrobium facilis, Hyphomicrobium denitrificans, Methylobacillus glycogenes, Methylobacterium extorquens, and Methylophilus methylotrophus is described. It could be demonstrated that vectors based on the broad-host-range plasmid pBBR1 could be transferred into these strains. Plasmid pBBR1KAN (3.9 kb), a kanamycin-resistant derivative of pBBR1, was suitable for transformation experiments in these methylotrophic bacteria. Transformation efficiencies up to 104transformants/μg plasmid pBBR1KAN were obtained. The broad-host-range plasmid pLA2917 was transferred into Hyphomicrobium species by a triparental mating. However, this plasmid was integrated into the genome of Hyphomicrobium spp. Plasmids pLA2917, pKT231, pSUP2021, pRZ705, and phage DNA could not be transferred in Hyphomicrobium spp. by electroporation under the conditions applied.Key words: Hyphomicrobium, transformation, methylotrophic bacteria, plasmid pBBR1, broad-host-range vector.
Stable transfection ofAcanthamoeba
Authors: Yin Jingwen , Henney Jr. Henry R.
Published: February 2010

The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate – DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 μg/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU – Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.Key words: Acanthamoeba, transfection, ubiquitin, promoter, vector, CAT assay.
Lack of genetic variability in French identified races of Plasmopara halstedii, the cause of downy mildew in sunflower Helianthus annuus
Authors: Roeckel-Drevet Patricia , Coelho Véronique , Nicolas Paul , Tourvieille Jeanne , Labrouhe Denis Tourvieille de
Published: February 2010

Downy mildew of sunflower (Helianthus annuus L.) incitated by Plasmopara halstedii is a potentially devastating disease. We report here the finding of two new races of P. halstedii and also two Apron35S fungicide-resistant isolates of race A. Using the random amplified polymorphic DNA (RAPD) technique as an initial screening for genetic variation within P. halstedii French races, genetic variation was not found between isolates within races 1, A, or B, and very few polymorphisms were distinguished between all French races known today.Key words: Plasmopara halstedii, race, genetic variability, RAPD.
Activités métaboliques et toxicité du dinoflagellé Alexandrium tamarense en culture à dialyse exposée à un milieu estuarien
Authors: Marsot P.
Published: February 2010

Metabolic activities and toxicity of the dinoflagellate Alexandrium tamarense were studied for a dialysis culture exposed to unenriched natural seawater and a batch culture grown in an enriched medium. The following observations were made on the dialysis culture compared with the batch culture: (i) Depending on the season period, the cell densities per litre were similar (≈ 10 × 106 – 14 × 106) or greater (66 × 106). (ii) During growth and stationary phases, cellular consumption of the nutrients (NO3− + NO2−) was much greater (5–50 times), (iii) During growth, the composition of the intracellular (IFAA) and extracellular dissolved free amino acid (EFAA) was characterized by the presence of ORN and LYS in the dialysis culture, which represent a substantial fraction of the IFAA pool of the dinoflagellate; a significant depletion of the IFAA was observed, but the recovery of Gln, Glu, and Arg cell concentration levels at the stationary phase was more important. (iv) During the growth phase, the maximum excretion rate of amino acids (fmol∙cell−1∙d−1) was 263 (in decreasing order of importance: Glu, Gin, Ser, Asp, Leu, Ala, G+T, and Orn) compared with 14,5. (v) During stationary phase, the reduction in excretion activity was twofold with Gln, Glu, Leu, and Orn while 9,2 fmol∙cell−1∙d−1 were observed in batch culture, (vi) Total toxin content was about 4 to 5 times higher (means: 95 and 75 fmol∙cell−1 during growth and stationary phases respectively), with greater concentrations in more potent derivates like saxitoxin and neosaxitoxin. This higher cell toxicity may be attributed to the particular growth conditions of the dialysis culture, which more closely approximate the natural environment, through diffusion processes involving a constant elimination of low molecular weight autoinhibitory metabolites and a more efficient nutrient uptake.Key words: Alexandrium tamarense, dinoflagellate, dialysis culture, phycotoxins, nitrogen metabolism.
The incidence of killer activity and extracellular proteases in tropical yeast communities
Authors: Abranches Jacqueline , Mendonça-Hagler Leda C. , Hagler Allen N. , Morais Paula B. , Rosa Carlos A.
Published: February 2010

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.Key words: killer yeasts, extracellular proteases, tropical yeast communities.
Polygalacturonase isolated from the culture of the psychrophilic fungus Sclerotinia borealis
Authors: Takasawa Toshihide , Sagisaka Keiko , Yagi Koichi , Uchiyama Kyoko , Aoki Atsushi , Takaoka Kyo , Yamamato Katsuhiro
Published: February 2010

A polygalacturonase was isolated from the culture medium of Sclerotinia borealis, a psychrophilic fungus that grows on lawn and wheat seedling under the snow in winter and induces the snow mold disease. Pectic acid was a better substrate of this enzyme than pectin when the activity was determined by measuring the reducing sugar produced. However, when the activity was measured by viscosity change, the viscosity of pectin decreased more rapidly than that of pectic acid. The results of viscosity change apparently indicate that the polygalacturonase catalyzes pectin hydrolysis as an endo-type enzyme. Highly methyl-esterified pectin was a poor substrate, as determined by measurements of reducing sugar production and viscosity change. It is suggested from the results that the methoxy group of pectin affects the polygalacturonase reaction. A reaction mechanism was proposed for the polygalacturonase reaction. Molecular mass of this enzyme was 40 kDa and its isoelectric point was pH 7.5. Optimum pH of the enzyme reaction was 4.5 and its optimum temperature was 40–50 °C. Thirty percent of the maximum activity was observed at 5 °C, but it was only slightly active above 60 °C. The activity was preserved for more than 2 years at 5 °C and pH 4.5, but it was lost when kept at room temperature overnight or heated at 50 °C for 30 min. The amino acid sequence of the N-terminal region of the psychrophilic polygalacturonase of Sclerotinia borealis is compared with those of polygalacturonases of mesophilic fungi. The function of this enzyme against the target plants is discussed with reference to the reaction of polygalacturonases of mesophilic fungi.Key words: polygalacturonase, pectin-hydrolyzing enzyme, psychrophilic fungi, snow mold disease, Sclerotinia borealis.
Structural features of ether lipids in the archaeobacterial thermophilesPyrococcus furiosus,Methanopyrus kandleri,Methanothermus fervidus, andSulfolobus acidocaldarius
Authors: Sprott G. Dennis , Agnew Brian J. , Patel Girishchandra B.
Published: February 2010

The ether lipids of several thermophilic archaea (archaeobacteria) were compared by negative-ion fast atom bombardment mass spectrometry. The major polar lipids in extracts of Pyrococcus furiosus were assigned as archaeol lipids (phosphatidylglycerol diether, m/z 805; phosphatidylinositol diether, m/z 893; and diglycosyl diether, m/z 975) and caldarchaeol lipids (diglycosyl phosphatidylglycerol tetraether, m/z 1778; and diglycosyl phosphatidylinositol tetraether, m/z 1866). The polar lipids of Methanopyrus kandleri were primarily glycolipids consisting of a series of archaeol lipids with one to six hexose units, composed primarily of mannose (mannose:glucose 9:1); phospholipids consisting of archaeol lipids (phosphatidylinositol diether; and a novel phosphatidylcholine diether, m/z 802.7), and phosphoglycolipids as minor caldarchaeol lipids (primarily diglycosyl phosphatidylglycerol tetraether). Methanothermus fervidus extracts contained archaeol lipids (phosphatidylinositol diether; diglycosyl diether; and acetyldiglycosyl diether, m/z 1016), and caldarchaeol lipids (glycosyl phosphatidylinositol tetraether, m/z 1704; diglycosyl phosphatidylinositol tetraether; and acetyldiglycosyl phosphatidylinositol tetraether, m/z 1907). Acetylation of a sugar residue occurred commonly in this thermophile and increased as cells entered the stationary growth phase. Lipid extracts of Sulfolobus acidocaldarius contained detectable amounts of archaeol and hydroxyarchaeol analogs of phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine lipids, in addition to the dominant caldarchaeol lipids already reported. All four thermophiles contained both archaeol and caldarchaeol lipids and phosphoinositol head groups, but no single structural entity uniquely separated their lipids from those found previously in mesophilic archaea. By contrast, extremely halophilic archaea appear to be distinguished from the thermophilic archaea by the presence of a major phosphatidylglyceromethylphosphate lipid.Key words: ether lipids, mass spectrometry, hyperthermophiles, extreme halophiles, Archaea.
An endoglucanase from the anaerobic fungusOrpinomyces joyonii: characterization of the gene and its product
Authors: Liu Jin-Hao , Selinger L. Brent , Hu You-Ji , Cheng Kuo-Joan , Beauchemin Karen A. , Moloney Maurice M.
Published: February 2010

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 °C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.Key words: endoglucanase, Orpinomyces joyonii, recombinant protein, expression.
Soil isolates of Pseudomonas spp. that utilize inositol phosphates
Authors: Richardson A. E. , Hadobas P. A.
Published: February 2010

Soil bacteria that utilize inositol hexaphosphate (IHP) were isolated from a range of soils using defined selection media. An analysis of 200 randomly selected isolates indicated that less than 0.5% of the culturable population of soil bacteria were capable of using IHP as a sole source of C and P. From a further 238 isolates obtained from enrichment culture, four unique organisms (identified by randomly amplified polymorphic DNA – polymerase chain reaction) were selected and characterized for their ability to specifically utilize IHP. These four organisms were putatively identified as either fluorescent Pseudomonas spp. (P. putida CCAR53 and CCAR59) or nonfluorescent Pseudomonas spp. (P. mendocina CCAR31 and CCAR60) as determined by partial DNA sequence analysis of 16S rRNA genes. The fluorescent Pseudomonas strains exhibited marked phytase activity and liberated up to 81% of the phosphate from IHP either in the absence or presence of arabinose as an additional C source. The nonfluorescent strains also exhibited an ability to liberate Pi from IHP but were effective only in the presence of added arabinose. Strains CCAR59 and CCAR60 could effectively utilize either Na-IHP or Ca-IHP at pH 7.0, whereas only strain CCAR59 could grow and utilize these substrates at pH 5.0.Key words: inositol hexaphosphate, soil phytate, phytase activity, Pseudomonas spp.
Use of Tn5-gusA5to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster ofPseudomonas syringaepv.glycinea
Authors: Palmer David A. , Bender Carol L. , Sharma Shashi B.
Published: February 2010

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized; however, the precise factors that regulate the production of COR and its precursors remain unclear. In the present study, a λ delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18–24 °C and significantly less expression at 14 and 30 °C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.Key words: transcriptional fusion, glucuronidase, gene expression, reporter gene.
Correlation ofPseudomonas aeruginosavirulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse
Authors: Woods D. E. , Lam J. S. , Paranchych W. , Speert D. P. , Campbell M. , Godfrey A. J.
Published: February 2010

The potential pathogenicity of a microorganism is a major concern for Health Canada evaluators, who will be processing new biotechnology products under the Canadian Environmental Protection Act. Potential pathogenicity is generally predicted by the results of animal pathogenicity studies. In an attempt to define surrogate data for an animal model, this study was initiated. Pseudomonas aeruginosa isolates from clinical and environmental sources were screened for their pilus type, serotype, lipopolysaccharide type, ability to evade host responses, and production of toxin A, exoenzyme S, elastase, phospholipase C, and total protease. The 50% lethal dose (LD50) of the same isolates was determined in the neutropenic mouse model of infection. An attempted correlation was drawn between each (or combinations) of the virulence determinants and the LD50. Stepwise linear regression showed that the presence of high levels of exoenzyme S in association with elastase or phospholipase C, or to a minor extent toxin A, was correlated with low numbers of bacteria required to elicit an LD50. No correlation between any of the other factors examined and virulence was detected. The data suggest that an in vitro high level of exoenzyme S production could be used as surrogate information for neutropenic mouse modelling; however, the levels of all of the extracellular enzymes should be considered when making such an assessment.Key words: virulence, exotoxins, exoenzyme S, clinical strains, environmental strains.
Morphological and chemical alterations inBotrytis cinereaexposed to the dicarboximide fungicide vinclozolin
Authors: Cabral Silvia M. J. C. S. , Cabral João P. S.
Published: February 2010

Treatment of actively growing Botrytis cinerea hyphae with micromolar concentrations of the dicarboximide fungicide vinclozolin resulted in significant alterations in the growth rate, morphology, and chemical composition of the cells. The addition of vinclozolin resulted in an immediate and severe reduction in the hyphal growth rate and a retardation in the emergence of the second germ tube. Cells treated with vinclozolin had a lower content of pool metabolites than control cells, and this difference increased with time of exposure to the fungicide. In contrast, vinclozolin-treated cells had a higher chitin concentration than control cells. These biochemical alterations were followed by the disorganization and clearing of cells, and by the appearance of dense and dark masses outside the hyphae, presumably composed of cell debris. Hyphae exposed to vinclozolin were more curved and branched and had shorter cells than the controls. The results indicate that vinclozolin causes a slow but generalized leakage of pool metabolites; this release precedes cell lysis and is not the result of a rapid and gross damage to the cytoplasmic membrane.Key words: vinclozolin, Botrytis cinerea, pool metabolites, membrane damage.
Interaction between poly-3-hydroxybutyrate-co-3-hydroxyvalerate and a denitrifying Pseudomonas strain
Authors: Biedermann J. , Schloe K. T. , Süßmuth R. , Owen A. J. , Gassner F.
Published: February 2010

In a laboratory-scale system, denitrification activity of a heterotrophic microbial starter culture changed when different lots of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) were used as the solid carbon source in the heterotrophic denitrification reactor. In this study, possible influences of physical and chemical properties of commercially produced P(HB-co-HV) (Biopol®) on biofilm formation and metabolic activity of a denitrifying starter culture were investigated. These parameters indicate the polymers' suitability for the application as the matrix substance in the bioreactor. No differences in microstructure were detected between the different lots of polymers. Growth inhibitory effects by chemical additives were found in the case of triacetine, which was included as a plasticizer in seven of eight tested lots. The amount of hydroxyvaleric acid in the polymer was not assumed to affect denitrification activity. Relevant differences could be detected regarding primary adhesion of the starter culture Pseudomonas sp. strain 2nIII. It showed good adsorption properties to hydrophobic substances with a dependence on precultivation conditions. Pseudomonas sp. strain 2nIII degraded poly-3-hydroxybutyrate acid homopolymer and P(HB-co-HV) copolymers but was unable to break up poly-3-hydroxyvaleric acid. A possible reason for these findings is the substrate specifity of the polyhydroxyalkanoate depolymerase.Key words: poly-3-hydroxybutyrate-co-3-hydroxyvalerate, drinking water denitrification, biofilm, microbial adsorption.
Pyridine degradation and heterocyclic nitrification by Bacillus coagulans
Authors: Uma B. , Sandhya S.
Published: February 2010

A gram-positive, pyridine-degrading microorganism identified as Bacillus coagulans has been isolated from contaminated soil by enrichment culture technique. Pyridine was used as sole source of carbon, nitrogen, and energy. Bacillus coagulans has a unique potential to reduce nitrogen from aromatic ring to ammonia and subsequently heterotrophically to nitrite and nitrate. The maximum degradation of pyridine was 94.1% within 72 h at 30 °C with a 7.57-h doubling time. The study suggests possible existence of aromatic degradation and heterotrophic nitrification in Bacillus coagulans.Key words: pyridine degradation, heterotrophic nitrification, Bacillus coagulans.
Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview
Authors: Liss Steven N. , Bicho Paul A. , Saddler John N.
Published: February 2010

Resin acids, a group of diterpenoid carboxylic acids present mainly in softwood species, are present in many pulp mill effluents and toxic to fish in recipient waters. They are considered to be readily biodegradable. However, their removal across biological treatment systems has been shown to vary. Recent studies indicate that natural resin acids and transformation products may accumulate in sediments and pose acute and chronic toxicity to fish. Several resin acid biotransformation compounds have also been shown to bioaccumulate and to be more resistant to biodegradation than the original material. Until recently, the microbiology of resin-acid degradation has received only scant attention. Although wood-inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive evidence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recently been described which are able to utilize dehydroabietic or isopimaric acids as their sole carbon source. There appears to be an unusually high degree of substrate specificity with respect to the utilization of abietane congeners and the presence of substituents. Pimaranes do not appear to be attacked to the same extent as the abietanes. This paper reviews the occurrence, chemistry, toxicity, and biodegradation of resin acids in relation to the biological treatment of pulp and paper mill effluents.Key words: resin acids, biodegradation, pulp mill effluents.
Murine macrophage elastolytic activity induced byAspergillus fumigatusstrains in vitro: evidence of the expression of two macrophage-induced protease genes
Authors: Rodriguez Eric , Boudard Frederic , Mallié Michele , Bastide Jean-Marie , Bastide Madeleine
Published: February 2010

The interaction between Aspergillus fumigatus conidia and murine macrophages of various origins was investigated. Cocultures were carried out between A. fumigatus strains and freshly isolated murine pulmonary alveolar macrophages or two murine macrophage cell-lines: murine alveolar cell-line MALU and murine astrocytoma cell-line J774. By measuring the variation of elastolytic activity in the coculture supernatants with two elastin substrates, we demonstrated that either viable or fixed A. fumigatus or C. albicans yeasts or nonspecific particles induced significant macrophage elastolytic activity. The effect of A. fumigatus supernatant or the purified A. fumigatus galactomannan suggested also the possible involvement of this polysaccharide in macrophage-protease gene expression, release, and activity in invasive aspergillosis. The effect of inhibitory compounds demonstrated the potential implication of a macrophagic metalloprotease and a macrophagic cysteine protease. RNA analysis allowed us to demonstrate the induction of expression of two macrophagic protease genes in stimulated macrophages. Two distinctive mechanisms appeared to be implicated in macrophage protease induction: nonspecific phagocytosis in the earliest times of the coculture and (or) specific galactomannan recognition after its gradual release by the mycelium.Key words: Aspergillus fumigatus, macrophages, proteases, invasive aspergillosis, galactomannan.
Electron microscopical observations and chemical analyses supporting Mn uptake in white rot degradedAlstoniaand pine wood stakes exposed in acid coniferous soil
Authors: Daniel Geoffrey , Nilsson Thomas , Volc Jindrich
Published: February 2010

Brownish-black spots and flecks several millimetres wide were frequently recognized on the surface and within nonpreservative-treated hardwood (Alstonia scholaris (R.Br.)) and softwood (Pinus sylvestris L.) test stakes placed in an acid forest soil. Energy-dispersive X-ray microanalysis in conjunction with transmission electron microscopy (TEM) and gross chemical analysis of wood using inductively coupled plasma spectrometry showed the flecks to be composed primarily of Mn, which was selectively removed from the surrounding soil. A similar uptake of Mn into wood stakes placed in presterilized acid soil was not noted, indicating the process resulted from biotic activity. Detailed TEM observations showed intrusion of Mn into the wood cell lumina and into areas of erosion, cavity formation, and decayed middle lamella in Alstonia and pine wood cells attacked by an unknown white rot decay fungus. Distinct zones of apparent delignification were also noted progressing across secondary cell walls and middle lamella regions of attacked cells, although it was unclear if the effect was caused by nonenzymatic attack by Mn, enzymatic attack by the fungus, or a combination of both. Mn is thought to play a major regulating role in both lignin depolymerization and mineralization in the presence of organic acids during white rot decay. Present observations also suggest that uptake of Mn into wood stakes during microbial degradation results from biotic activity and soil type and pH are of major significance.Key words: Mn, test stakes, electron microscopy, white rot decay, inductively coupled plasma spectrometry.
Sources of variation in isolation rate of Giardia lamblia cysts and their homogeneous distribution in river water entering a water treatment plant
Authors: Payment Pierre , Berte Aminata , Fleury Carole
Published: February 2010

The objective of this work was to determine if differences in the number of Giardia cysts measured in river water were due to the method itself, the analyst, or real differences in the distribution of these cysts in water. To minimize the methodological differences, centrifugation only was used as the primary concentration method. Differences were observed between results from different analysts and they were identified as technical errors. Once the method had been well established, cysts were found to be distributed homogeneously in the river water tested. Small differences were observed among samples collected sequentially at the same time, as well as for samples collected on different days or at different times on the same day. The differences reported in the literature in the number of Giardia cysts detected in water samples from the same site could be an artifact of the methods more than true differences in the counts.Key words: water, Giardia, parasite, methods.
Purification and characterization of a cytotoxin fromEnterobacter cloacae
Authors: Barnes Ana Isabel , Ortiz Cristina , Paraje María Gabriela , Balanzino Luis Eduardo , Albesa Inès
Published: February 2010

Leukotoxic activity was assayed in clinical isolates of Enterobacter cloacae. Two strains were selected out of 38 by their greater hemolytic activity in blood agar plates. Leukotoxin was purified by salt precipitation, dialysis, chromatography by gel filtration, and high pressure liquid chromatography (HPLC). Human leukocytes, when incubated with purified E. cloacae toxin, showed high percentages of death and lysis, with time and dose dependence. The chromatographic profile of gel filtration presented three protein peaks and toxic activity was detected in the second peak. After HPLC, leukotoxin coeluted with the hemolytic activity and both activities were detected only after 2-mercaptoethanol treatment. Coomassie-stained sodium dodecyl sulfate – polyarylamide gels showed a single band. This band was estimated to represent a protein of 13 300 Da on the basis of both sodium dodecyl sulfate – polyacrylamide gel electrophoresis and gel filtration chromatography.Key words: Enterobacter clocae, leukotoxin, molecular mass, cytotoxin, leukocytes.
Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase fromBartonella bacilliformis
Authors: Mitchell Samuel J. , Minnick Michael F.
Published: February 2010

We have cloned the inorganic pyrophosphatase gene (ppa) from the facultative intracellular pathogen Bartonella bacilliformis and characterized its encoded product. The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B. bacilliformis. The predicted protein encoded by ppa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22–23 kDa. The predicted B. bacilliformis pyrophosphatase (PPase) sequence is 53% identical and 85% similar to the E. coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catalytic active site. The isolated B. bacilliformis PPase exhibits an activity of 51 ± 2 μmol PO4released/(mg protein∙min) at 28 °C and pH 8, and is sensitive to inhibition by Ca2+. In keeping with other prokaryotic PPases, B. bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+(highest activity at 55 °C, relative activity = 80 ± 3% at pH 8). The cloned B. bacilliformis ppa is able to genetically complement a ppa−mutant strain of E. coli.Key words: Bartonella, invasion-associated locus, inorganic pyrophosphatase.
Influence of trophic conditions on exopolysaccharide production: bacterial biofilm susceptibility to chlorine and monochloramine
Authors: Samrakandi Moulay Mustapha , Roques Christine , Michel Georges
Published: February 2010

This study examines the controversial efficacy of chlorine and monochloramine against biofilms that differ in their extracellular polysaccharide (EPS) content. The results point out a net variability of bacterial biofilm susceptibility according to the nutrients present. Chlorine and monochloramine showed an equal biocidal activity on lactose medium-grown E. coli ATCC 10 536 and glycerol – ammonium nitrate medium-grown nonmucoid Pseudomonas aeruginosa biofilms. In contrast, the effect of monochloramine is greater compared with that of chlorine on E. coli and mucoid P. aeruginosa biofilms grown in sucrose and glycerol – ammonium nitrate media, respectively. In these culture conditions, treatment with 25 mg monochloramine/L for 2 h reduced culturable cells by 4.5 logs (99.997%) for E. coli and about 3 logs (99.87%) for mucoid P. aeruginosa while the similar treatment with chlorine reduced culturable cells in these biofilms by 2.2 logs (99.4%) and 1 log (10%), respectively. The decrease of chlorine disinfection efficacy on sucrose and glycerol – ammonium nitrate medium-grown biofilms is postulated to be linked to the higher polysaccharide production observed in these media. It seems likely that monochloramine produces a high leakage of material absorbing at 260 nm from sucrose medium-grown E. coli biofilm, which could indicate its better penetration into biofilms.Key words: biofilm, chlorine, monochloramine, E. coli, mucoid Pseudomonas aeruginosa.
Nematophin, a novel antimicrobial substance produced byXenorhabdus nematophilus(Enterobactereaceae)
Authors: Li Jianxiong , Chen Genhui , Webster John M.
Published: February 2010

A new antibiotic, nematophin, was isolated from strain BC1 of Xenorhabdus nematophilus and detected in all strains of X. nematophilus studied. Its structure is fully established as 3-indoleethyl (3′-methyl-2′-oxo)pentanamide by extensive spectroscopic study. The production of nematophin is affected by the strain type and culture conditions. The compound shows strong in vitro bioactivity against a series of fungal and bacterial species.Key words: nematophin, symbiotic bacteria, Xenorhabdus nematophilus, antibiotic.
Studies on the transformation of selected polychlorinated biphenyl congeners by Pseudomonas strain LB 400
Authors: Billingsley K. A. , Ward O. P. , Backus S. M.
Published: February 2010

Resting cells of Pseudomonas strain LB400 are known to transform polychlorinated biphenyls (PCBs) when the cells are previously grown on biphenyl. In this study, PCB transformation was also observed in resting cells grown on other substrates such as glucose and glycerol. The presence of PCB congeners in the growth medium increased the lag phase for the growth of cells on a biphenyl substrate but not on a glycerol substrate. Supplementation of the degradation medium with biphenyl dramatically decreased the rate of PCB congener transformation, while the presence of glycerol or glucose had little or no effect on PCB transformation rates. Removal rates with biphenyl-grown cells in the standard degradation medium for 2,4,2′,4′-tetrachlorobiphenyl, 2,4,5,2′,5′-pentachlorobiphenyl, and 2,3-dichlorobiphenyl were 1.06, 1.66, and 224 μmol/(L∙h), respectively. Relative rates of transformation of 2,3-dichlorobiphenyl by biphenyl-, glucose-, and glycerol-grown cells were 100:36:36 and were similar to the relative rates of transformation of 2,4,5,2′,5′-pentachlorobiphenyl (100:33:42). The presence of PCBs adversely affected cell viability of biphenyl-grown cells over a 48-h incubation period and may explain the decline observed in PCB conversion capacity over the same incubation period. A major objective of this study was to investigate the significance of using biphenyl as the carbon source for growth of Pseudomonas strain LB400 cells capable of PCB transformation. Our findings indicate that, whereas higher rates of transformation of PCBs are observed with biphenyl-grown cells, cells grown on other carbon sources retain PCB-transforming enzymes. In addition, it has been demonstrated that biphenyl inhibits transformation of PCBs by the organism, whereas glycerol or glucose does not.Key words: Pseudomonas strain LB400, polychlorinated biphenyls, degradation, biphenyl.
Bacterial genetic loci implicated in thePseudomonas putidaGR12-2R3 – canola mutualism: identification of an exudate-inducible sugar transporter
Authors: Bayliss Catherine , Bent Elizabeth , Culham Doreen E. , MacLellan Shawn , Clarke Anthony J. , Wood Janet M. , Brown Gerry L.
Published: February 2010

Pseudomonas putida GR12-2R3 promotes the emergence and growth of diverse plant species. Analyses of TnphoA insertion mutations are revealing bacterial characteristics pertinent to the plant–microbe interaction. Pseudomonas putida PG269 is a TnphoA insertion derivative of GR12-2R3 that expresses canola seed exudate-inducible alkaline phosphatase (PhoA) activity. It promoted the growth of canola roots, as well as strain GR12-2R3, and outgrew its parent when they were cocultured in the presence of canola roots or in liquid seed exudate medium. (In contrast, mutant PG126 failed to promote canola root growth and was outgrown by its parent strain.) The PhoA activity of strain PG269 was induced by glucosamine and other sugars; glucosamine inhibited the growth of strain GR12-2R3 and stimulated the growth of strain PG269. Strain PG269 contained two TnphoA insertions: seiA1:: TnphoA and seiB1:: TnphoA. Strain PG312, which contained only insertion seiA1:: TnphoA, shared all aspects of the PG269 phenotype, except the ability to outcompete strain GR12-2R3 during coculture. Insertion seiA1::TnphoA interrupted an open reading frame related in sequence to members of the MalF family of sugar transporter subunits. The PhoA-inducing fraction of canola seed exudate was hydrophilic, low in molecular weight, and heat stable. It cochromatographed with basic amino acids and amino sugars, and was inactivated by strains GR12-2R3 and PG269. Gene seiA may encode a subunit of an ABC transporter with broad specificity for glucose and related sugars whose expression can be induced by exudate sugars.Key words: Pseudomonas putida, canola, exudate, sugar transport, rhizobacterium.
On the relation of colony variants to the time dependency of colony formation during adaptive mutation ofEscherichia coliFC40
Authors: MacLeod Patricia R. , MacLeod Robert A.
Published: February 2010

When Escherichia coli FC40 formed adaptive Lac+revenants on a selective agar medium containing lactose as the carbon source, the colonies which accumulated over several days were of two readily distinguishable types. Colonies of both types appeared both early and late on the plates. Cells of colonies that appeared early and late on the plates, irrespective of the type, when grown in liquid medium and replated, all formed colonies on the selective medium within 48 h. Cells of each colony type gave rise to colonies of both types and attempts to isolate cells of each type in pure culture were unsuccessful. It was concluded that the presence of two colony types in the cultures plated did not contribute to the observed time dependency of colony formation during adaptive mutation. The proportions of the two colony types arising from cultures of the Lac+revertants varied from culture to culture.Key words: Escherichia coli FC40, variants, adaptive mutation, colony accumulation.
Characterization of Rhizobium from root nodules of leguminous trees growing in alkaline soils
Authors: Surange S. , Wollum II A. G. , Kumar N. , Nautiyal C. Shekhar
Published: February 2010

High temperature, pH, and salt stresses in tropical alkaline soils limit nodulation and dinitrogen fixation by strains of Rhizobium from the root nodules of nitrogen fixing trees (NFTs). This study was conducted to determine the variability among Rhizobium strains isolated from different NFTs in growth response to high temperature, pH, and salt concentrations. Variable response to increases in temperature, pH, and salt concentrations was observed. Rhizobium strain isolated from Albizia lebbek survived at 50 °C, while Rhizobium strains isolated from Sesbania formosa, Acacia farnesiana, and Dalbergia sissoo were well adapted to grow on pH 12.0. All the Rhizobium strains tolerated salt concentrations up to 5.0%. Strains were further characterized with respect to utilization of 27 carbon sources and for their effectiveness in substrate utilization at pH 7.0 and 9.0. Generally higher rates of O2 consumption were observed at pH 7.0 compared with pH 9.0.Key words: Rhizobium, leguminous trees, root nodules, stress tolerance.
A modified method for the enumeration of aerobic spore-forming bacteria
Authors: Barbeau Benoit , Boulos Lina , Desjardins Raymond , Coallier Josée , Prévost Michèle , Duchesne Daniel
Published: February 2010

An optimized method for cultivating aerobic spore-forming bacteria in drinking water is described. Such organisms offer the potential as an indicator of water treatment efficiency. The various parameters evaluated were recovery media, incubation, and pasteurization conditions. The optimal conditions consisted of pasteurization for 15 min at 75 °C, followed by membrane filtration and incubation for 24 h at 35 °C on a pad to which 1.4 mL trypticase soy broth had been added. Aerobic spore-forming bacteria were detected in large numbers (1.0 × 102 − 3.2 × 103 CFU/100 mL) in raw waters and daily samples showed that in natural waters they remained stable over the test period (3 weeks).Key words: aerobic spores, method, indicator.
Survival ofEscherichia coliexposed to visible light in seawater: analysis ofrpoS-dependent effects
Authors: Gourmelon M. , Pommepuy M. , Touati D. , Cormier M.
Published: February 2010

We investigated the effect of visible light on Escherichia coli in seawater microcosms. Escherichia coli lost its ability to form colonies in marine environments when exposed to artificial continuous visible light. Survival of illuminated bacteria during the stationary phase was drastically reduced in the absence of the σsfactor (RpoS or KatF) that regulates numerous genes induced in this phase. In the stationary phase, double catalase mutants katE katG and mutants defective in the protein Dps (both catalase and Dps are involved in resistance to hydrogen peroxide (H2O2)), were more sensitive to light. In the exponential phase, a mutation in oxyR, the regulatory gene of the adaptive response to H2O2, increased sensitivity to light, further suggesting that deleterious effects might be associated with H2O2production. However, in the stationary phase, the katE katG dps mutant was considerably more resistant to visible light than the rpoS mutant, suggesting rpoS-dependent protection against deleterious effects other than those related to H2O2. The deleterious action of visible light was less important when the salinity decreased. In freshwater, rpoS and katE katG dps mutants did not show a drastic difference in sensitivity to light suggesting that osmolarity sensitizes E. coli to those deleterious effects of visible light that are unrelated to H2O2.Key words: Escherichia coli, stationary phase, RpoS, visible light, seawater.
Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2
Authors: García Pilar , Suárez Juan Evaristo , Bascarán Victoria , Rodríguez Ana
Published: February 2010

Random Sau3AI DNA fragments from the temperate Lactobacillus bacteriophage A2 were cloned into the promoter-probe plasmid pGKV210. Seven DNA fragments with promoter activity were selected, after transformation of Escherichia coli and Lactococcus lactis, subsp. lactis, through the chloramphenicol resistance they conferred to the corresponding clones. The seven promoters were functional in Lactobacillus casei. Their strength was analysed by measuring the levels of chloramphenicol resistance and chloramphenicol acetyltransferase activity induced in each host. The nucleotide sequences of these fragments were determined and primer extension analysis was used to locate the initiation site of transcription from each promoter in E. coli. The promoters contained −10 and −35 regions similar to the consensus sequences of E. coli and Lactobacillus promoters.Key words: bacteriophage, Lactobacillus, promoter.