摘要:

TwoBacillus thuringiensisvar.kurstakiHD-1 mutants, twoBacillus thuringiensisvar.israelensisHD-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 ofcryI- andcryIV-type genes was performed in a polymerase chain reaction usingcryIandcryIV-specific oligonucleotide primers. Thecry-type genes under study consisted ofcryIA(a),cryI(A)b,cryI(A)c,cryIB, andcryIV. Presence or absence of thecryI- andcryIV-type genes was further confirmed by Southern blotting followed by hybridization with specificcryIandcryIVgene fragments. A genetically modified strain ofB.thuringiensisvar.kurstakiHD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to containcryIA(a),cryIA(b), andcryIA(c) genes. AnotherkurstakiHD-1 mutant, called NGK-13 and obtained following treatment withN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost thecryIA(b) gene while retaining thecryIA(a) andcryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant ofB.thuringiensisvar.israelensis(Bti) HD-500, obtained following treatment with MNNG containedcryIV-typegenes. NGI-22, an oligosporogenous–acrystalliferous mutant ofBtiHD-500, contained nocryI- norcryIV-typegenes. The rice grain dust isolate BT-285 contained thecryIA(a) andcryIA(c) genes. Isolate BT-14 contained only thecryIA(c) gene, whereas isolate BT-209 containedcryIA(a),cryIA(b), andcryIBgenes. Isolate BT-205 contained nocryI- norcryIV-type genes.Bacillus thuringiensismutants and natural isolates shown to containcryI-type genes were tested for their insecticidal activities in a series of bioassays againstHyphantria cuneaDrury (Lepidoptera: Arctiidae). AllcryI-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.

ISSN:0008-4166    

DOI:10.1139/m97-057

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

The Scholander pressure bomb system, which expresses vascular plant sap, was compared with the trituration method, in which roots are surface disinfested and triturated, for recovery of endophytic bacteria. The two methods were compared for recovery of indigenous and introduced endophytes from roots of several plant genera. The pressure bomb method was acceptable for routine recovery of endophytes from cotton (Gossypium hirsutum), soybean (Glycine max), and bean (Phaseolus vulgaris), but owing to tissue collapse under pressure, the method did not work reliably for cucumber (Cucumis sativa) or tomato (Lycopersicon esculentum) seedlings. High bacterial densities on the root surface, experimentally obtained by dipping cotton roots into a suspension ofEnterobacter asburiaeJM22 immediately prior to processing, did not affect the population densities of recovered indigenous endophytic bacteria by the pressure bomb technique but resulted in increased bacterial densities for the trituration method. Internal populations of JM22 following application as a seed treatment were statistically equivalent with the trituration and pressure bomb techniques. Analysis of taxonomic diversity of a group of indigenous endophytes recovered with the trituration and pressure bomb techniques indicated some differences between the two groups. The total number of bacterial genera and species recovered was greater using the pressure bomb method. Gram-positive species, such asBacillusspp., were more frequently isolated with the trituration method than with the pressure bomb method.Agrobacterium radiobacterand less common species were more often isolated using the pressure bomb technique.Pseudomonasspp. andPhyllobacteriumspp. were recovered with equal frequencies using both techniques. These results suggest that the two techniques sample two different internal habitats available for colonization by endophytic bacteria, i.e., the trituration method recovering mainly endophytes residing in the root cortex and the pressure bomb method detecting vascular colonists. A combination of both methods is recommended for understanding the full pattern of internal plant colonization by endophytic bacteria.Key words: endophytic bacteria, Scholander pressure bomb, isolation method, cotton.

ISSN:0008-4166    

DOI:10.1139/m97-058

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

A polygalacturonase was isolated from the culture medium ofSclerotinia 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 ofSclerotinia borealisis 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.

ISSN:0008-4166    

DOI:10.1139/m97-059

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Pseudomonas marginalisis an important postharvest pathogen capable of causing soft rot in a wide variety of harvested fruits and vegetables. Following transposon mutagenesis, we isolated two groups ofP.marginalisCY091 mutants deficient in production of pectate lyase (Pel) and soft-rot pathogenicity in plants. The first group, designated Pel−, was caused by the insertion of Tn5into apelstructural gene, and the second group, designated LemA−, was caused by the insertion of Tn5into a regulatory locus corresponding to thelemAgene previously identified in other Gram-negative bacteria. The LemA−mutants also exhibited alteration in colony morphology and showed deficiency in production of protease (Prt). A cosmid clone pCIC carrying theP.marginalis lemAgene was isolated and characterized. pCIC was capable of restoring Pel production and soft-rot pathogenicity in LemA−mutants ofP.marginalisandPseudomonas viridiflava, indicating that the function oflemAgene in these two pseudomonads was similar and interchangeable. Using MudI-mediated mutagenesis, we isolated a third group ofP.marginalismutants deficient in production of Pel, Prt, and soft-rot pathogenicity. Mutants in this group (designated GacA−1) contained an insertion of MudI in a locus corresponding to thegacAgene ofP.viridiflava. Like LemA−mutants, GacA−mutants also exhibited alteration in colony morphology and showed deficiency in production of Pel and Prt. However, GacA−mutants produced much lower levels of levan and fluorescent pyoverdine siderophore than the wild type and LemA−mutants. These results provide the first genetic evidence thatP.marginalisproduces a single alkaline Pel for maceration of plant tissue and demonstrate that production of Pel, Prt, levan, and pyoverdin by this bacterium is mediated by the two-componentlemA/gacAgene system.Key words: two-component regulators, pectate lyase, protease, levan, pyoverdin.

ISSN:0008-4166    

DOI:10.1139/m97-060

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

The effect of carbon source on the levels of three (1 → 3)-β-glucanases and a (1 → 6)-β-glucanase in the culture filtrates of the filamentous fungusAcremonium persicinumwas investigated. All four enzymes were produced during growth of the fungus on (1 → 3)-, (1 → 6)-, and (1 → 3)(1 → 6)-β-glucans as well as β-linked oligoglucosides. However, only one (1 → 3)-β-glucanase and the (1 → 6)-β-glucanase were detected during growth on a range of other carbon sources including glucose, carboxymethylcellulose, and the α-glucan pullulan. The presence of glucose in the medium markedly decreased the production of all four glucanases, although the concentration required to effect complete repression of enzyme levels varied for the different enzymes. Similar repressive effects were also observed with sucrose, fructose, and galactose. The most likely explanations for these observations are that the synthesis of the (1 → 6)-β-glucanase and one of the (1 → 3)-β-glucanases is controlled by carbon catabolite repression, while the remaining two (1 → 3)-β-glucanases are inducible enzymes subject to carbon catabolite repression.Key words: (1 → 3)-β-glucanase, (1 → 6)-β-glucanase,Acremonium persicinum, regulation of synthesis, fungal β-glucanases.

ISSN:0008-4166    

DOI:10.1139/m97-061

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Two isolates of the acaropathogenic fungusHirsutella thompsonii(Nos. 255 and 414), andHirsutella necatrix, were able to produce and excrete chitinolytic enzymes. A chitobiase of > 205 kDa was excreted by all fungi and a chitobiase of 112 kDa only by isolate 414. An endochitinase of 162 kDa was excreted by isolate 414 and two endochitinases of 66 and 38 kDa were excreted by isolate 255. BothH.thompsoniiisolates produced chitinolytic enzymes only under inducible conditions, in the presence of colloidal chitin as the sole source of carbon.Hirsutella necatrixproduced a chitobiase constitutively when grown in the presence of glucose. In addition to chitinolytic enzymes, theH.thompsoniiisolates excreted proteolytic activities, including elastase, as well as α-esterase and α-amylase activities.Hirsutella necatrixwas unable to use casein, milk powder, or elastin as the sole carbon source. The acaropathogenicity of these isolates was assayed on the carmine spider mite (Tetranychus cinnabarinus). Isolates 414 and 255 andH.necatrixkilled ca. 80, 35, and 15%, respectively, of the infected mites. The role of chitinolytic and other enzymatic activities in the acaropathogenicity of these fungi is discussed.Key words: acaropathogenic fungi,Hirsutella, chitobiase, endochitinase, α-amylase.

ISSN:0008-4166    

DOI:10.1139/m97-062

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

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

ISSN:0008-4166    

DOI:10.1139/m97-063

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Total DNAs from nine strains ofAcetobacter xylinum, two strains ofAcetobacter aceti, and oneAcetobacter pasteurianusstrain were examined for the extent of digestion by various restriction endonucleases. The majority of the endonucleases cleaved the total DNAs with a frequency expected from the number of sites present in DNA sequences deposited in the GenBank data base. However, the restriction enzyme digestions identified two different genomic DNA modifications inAcetobacter. One sequence-specific modification protected total DNAs from seven of theA.xylinumstrains against cleavage byEcoRI (GAATTC). Digestion of total DNAs fromA.xylinumATCC 10245 (DNA not cut byEcoRI) and the closely relatedA.xylinumNRCC 17005 (DNA cut byEcoRI) withTsp509I (AATT) revealed differences in restriction frequencies that indicated methylation of the first or second adenine within GAATTC. Another sequence-specific modification rendered total DNAs from all the 12 strains recalcitrant to digestion byHinfI. The latter modification indicated that species of the genusAcetobactercontain a solitary DNA methyltransferase that probably methylates adenine in GANTC.Key words:Acetobacter, genomic DNA, modifications,EcoRI,HinfI.

ISSN:0008-4166    

DOI:10.1139/m97-064

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

A plaque technique that uses antibiotic-resistant bacteria growing on antibiotic-containing agar for the assay lawn resulted in significantly better recovery of bacteriophages P1 ofEscherichia coliand F116 ofPseudomonas aeruginosafrom nonsterile soil than standard membrane filtration or centrifugation techniques. Adsorption of the phages on soil particles appeared to be involved in their recovery and survival in soil.Key words: bacteriophages, soil, antibiotic-resistant bacteria, enumeration, filtration, centrifugation.

ISSN:0008-4166    

DOI:10.1139/m97-065

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

The ether lipids of several thermophilic archaea (archaeobacteria) were compared by negative-ion fast atom bombardment mass spectrometry. The major polar lipids in extracts ofPyrococcus furiosuswere assigned as archaeol lipids (phosphatidylglycerol diether,m/z805; phosphatidylinositol diether,m/z893; and diglycosyl diether,m/z975) and caldarchaeol lipids (diglycosyl phosphatidylglycerol tetraether,m/z1778; and diglycosyl phosphatidylinositol tetraether,m/z1866). The polar lipids ofMethanopyrus kandleriwere 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/z802.7), and phosphoglycolipids as minor caldarchaeol lipids (primarily diglycosyl phosphatidylglycerol tetraether).Methanothermus fervidusextracts contained archaeol lipids (phosphatidylinositol diether; diglycosyl diether; and acetyldiglycosyl diether,m/z1016), and caldarchaeol lipids (glycosyl phosphatidylinositol tetraether,m/z1704; diglycosyl phosphatidylinositol tetraether; and acetyldiglycosyl phosphatidylinositol tetraether,m/z1907). Acetylation of a sugar residue occurred commonly in this thermophile and increased as cells entered the stationary growth phase. Lipid extracts ofSulfolobus acidocaldariuscontained 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.

ISSN:0008-4166    

DOI:10.1139/m97-066

出版商:NRC Research Press    

年代:1997

数据来源: NRC