摘要:

Megasphaera cerevisiaeis a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates ofM. cerevisiaewere obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46 000, 45 000, and 43 000 for three, two, and two isolates, respectively. When mouse antiserum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination byM. cerevisiaebelonging to any serogroup.Key words: beer spoilage bacteria,Megasphaera cerevisiae, monoclonal antibodies, surface proteins, serogroups.

ISSN:0008-4166    

DOI:10.1139/w99-123

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

The chemical structure of cell walls and fractions ofVerticillium fungicola,a pathogen ofAgaricus bisporus,as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.Key words: cell walls, polysaccharides,Verticillium fungicola.

ISSN:0008-4166    

DOI:10.1139/w99-120

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungusPhanerochaete chrysosporiumstrain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT·L-1. The presence of 1% (w/v) Tween 80, at 20 mg·L-1TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth ofP. chrysosporiumwithout any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80.Key words: 2,4,6-trinitrotoluene, TNT, surfactant, white-rot fungus,Phanerochaete chrysosporium, lignin peroxidase, manganese peroxidase.

ISSN:0008-4166    

DOI:10.1139/w99-126

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

p-Cresol was repeatedly detected as a trace metabolite in anaerobic slurry reactors treating 2,4,6-trinitrotoluene (TNT)-contaminated soils. This study shows thatp-cresol was not a metabolite of the anaerobic degradation of TNT, by using a combination of analytical techniques and13C-labelled TNT. Instead,p-cresol, an intermediate in the degradation pathway of some amino acids, was shown to be inhibited by TNT and its metabolites. The range and persistence of inhibition top-cresol microbial degradation decreased with the level of amino-substitution of the derivatives. This explains whyp-cresol accumulated within the TNT-treating anaerobic bioslurry, as it could not be further biodegraded in the presence of TNT.Key words:p-cresol, bioremediation, trinitrotoluene, inhibition, metabolites.

ISSN:0008-4166    

DOI:10.1139/w99-124

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

A virulent strain of pigeon pea wilt pathogen was isolated from wilted pigeon pea plants and was identified asFusarium oxysporumf. sp.udum. Many bacterial cultures showing antagonism to the pathogen were isolated from various ecological niches. When tested under pot and field conditions, development of fusarial wilt symptoms was prevented in pigeon pea seeds treated with one such antagonist,Bacillus brevis. A formulation ofB. breviswith vermiculite as a carrier had a shelf life of at least 6 months.Bacillus brevisproduced an extracellular antagonistic substance which induced swelling of the pathogen's hyphal tips, and cells were bulbous and swollen with shrunken and granulated cytoplasm. The antagonistic substance also inhibited germination of conidia, and was fungicidal to the vegetative mycelia of the pathogen. Comparison of the properties of our antagonistic substance with that of known antibiotics produced byB. brevissuggests that our antagonistic substance is a novel compound. The observations reported here indicate that this strain ofB. brevismay have potential as a biocontrol agent against fusarial wilt in pigeon pea.Key words: antibiosis,Bacillus brevis, biological control, fusarial wilt, pigeon pea.

ISSN:0008-4166    

DOI:10.1139/w99-109

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium.Key words: consortium, catabolic gene, diclofop-methyl, 16S rDNA, FISH, SCLM.

ISSN:0008-4166    

DOI:10.1139/w99-129

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Fusariumspp. are ubiquitous fungi infecting cereals and grains, and therefore constitute a major problem for agriculture. Their trichothecene metabolites, and in particular deoxynivalenol and its 3-acetylated derivative, are the mycotoxins involved. The major metabolite produced byFusarium culmorumis 3-acetyldeoxynivalenol. Studies in vivo withFusarium culmorumhave established that its tricyclic intermediate, isotrichodermin, is a major biosynthetic precursor, which is hydroxylated at position 15 to give 15-deacetylcalonectrin, prior to being converted to the product. In a preliminary in vitro investigation of the cell-free system involved in this transformation, we suggested that cytochrome P450 enzymes are not involved. In this paper, the isotrichodermin-15-hydroxylase from the microsomal fraction ofFusarium culmorumwas solubilized and partially purified (60 fold). Our studies with cofactors indicate that this enzyme is a flavoprotein, and the inducers tested highly indicate that indeed the hydroxylase is not attached to cytochrome P450. This is particularly interesting, since the only other enzyme catalyzing the same reaction isolated fromFusarium sporotrichiodesis attached to cytochrome P450.Key words: trichothecene,Fusarium culmorum, cell-free system, isotrichodermin, 15-deacetylcalonectrin, flavoprotein.

ISSN:0008-4166    

DOI:10.1139/w99-116

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

To establish an efficient screening system for differentially expressed genes of a phytopathogenic fungusColletotrichum lagenarium,we constructed an equalized (normalized) cDNA library fromC.lagenariumand used this library for differential screening. For the isolation of genes involved in infection-related developments of conidia, conidia undergoing appressorium differentiation were selected as the source of materials for construction of the cDNA library. The equalization of cDNA was performed twice using a kinetic method, and the products were cloned into a plasmid vector. Colony hybridization with nine probes of different abundance showed a reduction in abundance variation from at least 276-fold in the original library to 10-fold in the equalized cDNA library, which demonstrated that the cDNA was successfully equalized. By differential hybridization of 1900 cDNA clones in the equalized cDNA library and RNA blot analysis of candidate clones, we identified 11 independent cDNA clones, designatedCAD1throughCAD11, that were expressed in appressorium-differentiating conidia, but not in vegetative mycelia. The transcripts ofCAD1andCAD2hardly accumulated in preincubated conidia, whereas those ofCAD3andCAD4accumulated highly and slightly, respectively. The amount of the fourCADtranscripts increased at the early stage of the appressorium formation process. Sequence analysis ofCAD1revealed thatCAD1would encode for 101 amino acid polypeptides, which showed homology to metallothioneins. Deduced amino acid sequence ofCAD2would encode 278 amino acid polypeptides, and showed high homology to genes in aflatoxin, and sterigmatocystin gene clusters ofAspergillus parasiticusandA. nidulans, respectively.Key words: equalized cDNA library, differential screening,Colletotrichum lagenarium, appressorium formation,CADgenes.

ISSN:0008-4166    

DOI:10.1139/w99-119

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27°C, complete virus inactivation took place in eight days. The temperature was then shifted to 4°C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle.Key words: poliovirus, MS-2, groundwater, virus inactivation, virus reactivatio

ISSN:0008-4166    

DOI:10.1139/w99-128

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

A key step which involves the cyclization of &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopenicillin N synthase (IPNS). In this study, an IPNS gene fromStreptomyces lipmaniiNRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed inEscherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS fromS. lipmanii16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Futhermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.Key words: isopenicillin N synthase, &bgr;-lactam antibiotics, secondary metabolism, consensus primers.

ISSN:0008-4166    

DOI:10.1139/w99-127

出版商:NRC Research Press    

年代:2000

数据来源: NRC