摘要:

Pichia kluyveri, a sexual ascomycetous yeast from cactus necroses and acidic fruit, is divided into three varieties. We used physiological, RAPD, and AFLP data to compare 46P. kluyveristrains collected worldwide to investigate relationships among varieties. Physiology did not place all strains into described varieties. Although the combined AFLP and RAPD data produced a single most parsimonious tree, separate analysis of AFLP and RAPD data resulted in significantly different trees (by the partition homogeneity test). We then compared the distribution of strains per band to an expected distribution. This suggested we could separate both the AFLP and RAPD datasets into bands from rapidly and slowly changing DNA regions. When only bands from slowly changing regions (from each dataset) were included in the analysis, both the RAPD and AFLP datasets supported a single tree. This second tree did not differ significantly from the cladogram based on all of the DNA data, which we accepted as the best estimate of the phylogeny of these yeast strains. Based on this phylogeny, we were able to demonstrate the strong influence of geography on the population structure of this yeast, confirm the monophyly of one variety, question the utility of maintaining another variety, and demonstrate that the physiological differences used to separate the varieties did not do so in all cases.Key words: RAPD, AFLP, yeast, data homogeneity,Pichia kluyveri.

ISSN:0008-4166    

DOI:10.1139/w00-092

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Heat shock protein 80 (Hsp80) ofNeurospora crassa, a member of the stress-90 protein family, is a cytosolic molecular chaperone that interacts directly with Hsp70 to form a hetero-oligomeric complex. The complete nucleotide sequence of the gene encoding this protein, along with the 5'- and 3'-flanking DNA, is reported. The coding sequence is interrupted by two introns, 61 and 30 nucleotides, respectively, in length. The deduced amino acid sequence corresponds to a 695-residue polypeptide with a calculated molecular mass of 78 894 Da and an average pI of 4.94. Primer extension experiments demonstrated two transcription start sites, a major and a minor one. No sequence motifs resembling the standard eukaryotic heat shock elements were evident in the putative promoter region. Immunoblot analysis showed Hsp80 protein to be present in the mature, dormant conidia, while thehsp80 transcripts were not detected. Both the transcripts and the protein were present in the germinating conidia in the absence of externally applied stress.Key words: Hsp90, filamentous fungi, sequence, conidia, germination.

ISSN:0008-4166    

DOI:10.1139/w00-095

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Mitochondria isolated from streptomycin-resistant (Sr) protoplasts ofPhytophthora parasiticawere transferred into chloramphenicol-resistant (Cpr) protoplasts ofP. parasiticaorPhytophthora capsiciwith an average successful rate of 1.7 × 10-4, using a selective medium containing streptomycin. No colonies appeared when self-fusion products of donor mitochondria or recipient protoplasts were exposed to the selective medium. Mitochondria isolated from Cprprotoplasts ofP. capsiciwere also transferred into Srprotoplasts ofP. parasiticawith a similar success rate using a selective medium containing chloramphenicol. Zoospores produced by the Cpr+Srintraspecific mitochondrial hybrid gave rise to Srand Cpr+Srcultures. The second generation zoospores produced by Srand Cpr+Srcultures also gave rise to Srand Cpr+Srcultures, suggesting the possible occurrence of fusion between some of the Cprmitochondria and Srmitochondria, and the displacement of non-fused Cprmitochondria in the receptor protoplast by the donor Srmitochondria. Zoospores produced by the interspecific mitochondrial hybrid gave rise to Cpr, Sr, Cpr+Sr, and Cps+Sscultures. The second generation zoospores produced by Cpr+Sror Srcultures also gave rise to the same four types of cultures, suggesting the existence of residual antibiotic-sensitive mitochondria (Cps+Ss) in the parental isolates and the random distribution of Cpr, Sr, and Cps+Ssmitochondria during asexual reproduction. Results suggest that the phenotype of antibiotic resistance / sensitivity was the end result of the interactions among the three types of mitochondria.Key words: mitochondrial transplantation, mitochondrial hybrid, antibiotic resistance,Phytophthora parasitica, Phytophthora capsici

ISSN:0008-4166    

DOI:10.1139/w00-093

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Methanosphaera stadtmanae(DSM 3091) is a methanogen that requires H2and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420oxidoreductase activity. The NADP+:F420oxidoreductase, the enzyme which catalyses the electron transfer between NADP+and F420in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420reduction was 6.0, and 8.5 for NADP+reduction. During the purification process, it was noted that precipitation with (NH4)2SO4increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme fromM. stadtmanaepossessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of &agr;, &bgr;, and &ggr; subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. TheKmvalues were 370 µM for NADP+, 142 µM for NADPH, 62.5 µM for F420, and 7.7 µM for F420H2. These values were different from theKmvalues observed in the cell-free extract.Key words: methanogen, NADP:F420oxidoreductase, NADP reductase, F420, NADP+.

ISSN:0008-4166    

DOI:10.1139/w00-090

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Keratinase is a serine protease produced byBacillus licheniformisPWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficientB. subtilisstrain. Studies presented herein demonstrate thatB. licheniformisCarlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes fromB. licheniformisstrains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that thekerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression ofsubE-encoded subtilisin fromB. subtilis(78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficientB. subtilisstrain. While strain PWD-1 and thekerAprotease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.Key words: keratin hydrolysis,Bacillus, subtilisin, keratinase.

ISSN:0008-4166    

DOI:10.1139/w00-085

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

In this study, we investigated the size and structure of autotrophic ammonia oxidizer (AAO) communities in the groundwater of a contamination plume originating from a mill-tailings disposal site. The site has high levels of dissolved N from anthropogenic sources, and exhibited wide variations in the concentrations of NO3-and NH3+ NH4+. Community structures were examined by PCR-DGGE targeting 16S rDNA with band excision and sequence analysis, and by analysis ofamoAfragment clone libraries. AAO population sizes were estimated by competitive PCR targeting the geneamoA, and correlated significantly with nitrate concentration. Most samples revealed novel diversity in AAO 16S rDNA andamoAgene sequences. Both 16S rDNA andamoAanalyses suggested that all samples were dominated byNitrosomonassp.,Nitrosospirasp. being detected in only 3 of 15 samples. This study indicated numerical dominance ofNitrosomonasoverNitrosospirain groundwater, and suggests that groundwater ammonia oxidizers are more similar to those dominating freshwater sediments than bulk soil.Key words:Nitrosomonas,Nitrosospira, nitrification, groundwater,amoA, 16S.

ISSN:0008-4166    

DOI:10.1139/w00-099

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Three composts were tested for their ability to suppress root and stem rot caused by the soil borne fungal pathogenFusarium oxysporumf. sp.radicis-cucumerinum(FORC) on cucumber. Two of the composts were prepared from separated dairy solids either by windrow (WDS) or vermicomposting (VMC) while the third, obtained from International Bio-Recovery (IBR), was prepared from vegetable refuse using aerobic digestion. Three sets of potting mixes were prepared by mixing the composts with sawdust at varying ratios, and seeded with cucumber cv. Corona. After 14 days of growth in the greenhouse, inoculum of FORC (20 mL of 5 × 106micro-conidia per mL) was applied to each pot at three different times (14, 21, and 35 days). In unamended inoculated pots, the pathogen caused stunted growth and reduced flowers. Amendment of WDS in the potting mix suppressed these symptoms, while VMC and IBR had no effect. All three composts reduced the FORC colony forming units (cfu) at the end of the experiment (10 weeks). There was a large increase of fluorescent bacteria near the vicinity of roots particularly in WDS amended potting mixes. When water extracts of the composts were plated onto acidified potato dextrose agar (APDA), only IBR contained a potent thermostable inhibitor to FORC. This inhibitor was removed by activated charcoal but was not partitioned into petroleum ether at acid, basic, or neutral pH. Inhibition of FORC by IBR was not due to electrical conductivity or trace elements in the compost. Contrasting effectiveness of the WDS and VMC made from the same waste suggests that composting method can influence the disease suppression properties of the finished compost.Key words:Fusarium oxysporumf. sp.radicis-cucumerinum, biological control

ISSN:0008-4166    

DOI:10.1139/w00-082

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

This is the first detailed report of xylanolytic activity inThermusstrains. Two highly thermophilic xylanolytic bacteria, very closely related to non-xylanolyticT. thermophilusstrains, have been isolated from the hottest zones of compost piles. Strain X6 was investigated in more detail. The growth rate (optical density monitoring) on xylan was 0.404·h-1at 75°C. Maximal growth temperature was 81°C. Xylanase activity was mainly cell-bound, but was solubilized into the medium by sonication. It was induced by xylan or xylose in the culture medium. The temperature and pH optima of the xylanases were determined to be around 100°C and pH 6, respectively. Xylanase activity was fairly thermostable; only 39% of activity was lost after an incubation period of 48 h at 90°C in the absence of substrate. XylanolyticT. thermophilusstrains could contribute to the degradation of hemicellulose during the thermogenic phase of industrial composting.Key words:Thermus, thermophilic aerobic bacteria, xylanase, thermostable enzyme, comp

ISSN:0008-4166    

DOI:10.1139/w00-075

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Pea plants grown in the field were used to study the natural incidence of endophytic bacteria in the stem. Eleven pea cultivars at the flowering stage were screened for the presence of endophytic bacteria using a printing technique with surface disinfested stem cross-sections on 5% Trypticase Soy Agar (TSA). Five cultivars showed colonization. Cultivar Twiggy showed the highest and most consistent colonization and was further investigated. Stems of cv. Twiggy at the pod stage were analyzed for endophytic bacterial types and populations. Cross-sections of surface disinfested stems were printed on 5% TSA. Endophytic bacterial populations decreased from the lower to the upper part of the stem. One section from the third and the fourth internode was surface disinfested, homogenized, and spiral plated on the media 5% TSA, R2A, and SC (Davis et al. 1980). Over a series of 30 samples, 5% TSA gave significantly better recovery of bacterial endophytes compared with R2A and SC media. For most stems, populations ranged from 104to 105CFU/g except in one of the field blocks in which endophyte populations were uniformly higher. Comparison of colony counts by spiral plating and printing showed a positive correlation. The most frequently recovered bacterial types werePantoea agglomeransandPseudomonas fluorescens. Less frequently isolated werePseudomonas viridiflavaandBacillusmegaterium.Key words: endophytic bacteria, pea, stem colonization, cultivar screening, biodiversity.

ISSN:0008-4166    

DOI:10.1139/w00-098

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

Chitin production byCunninghamella elegans(IFM 46109) was studied with a two-level full factorial design, varying time of cultivation and the concentration of D-glucose, L-asparagine, and thiamine in the culture medium. The material extracted was characterized by infrared and NMR spectroscopy. The highest chitin yield, 28.8%, was comparable with the highest in the literature and was obtained with a medium containing 60 g·L-1of glucose, 3 g·L-1of asparagine, and 0.008 mg·L-1of thiamine. Increasing the time of cultivation from 24 h to 72 h did not affect chitin production. The three factors showed significant positive effects on chitin production, without interactions between them.Key words: chitin, chitosan, zygomycetes,Cunninghamella elegans.

ISSN:0008-4166    

DOI:10.1139/w00-086

出版商:NRC Research Press    

年代:2000

数据来源: NRC