摘要:

We found that Congo red agar allows identification of sporulation-deficientBacillus anthracis. Using Congo red agar, we isolated an asporogenic derivative of the protective antigen-producing strainB.anthracisdeltaSterne-1(pPA102). Polymerase chain reaction and Southern hybridization analyses of DNA from the asporogenic mutant revealed that a deletion was present inspoOA,an essential gene for the initiation of sporulation. The deletion also encompassed thespoIVBhomologue and a portion of therecNhomologue. The avirulentspoOAstrain deltaSterne-1(pPA102)CR4 is suitable for the safe production of protective antigen without endospore contamination of the vaccine production facility.Key words:Bacillus anthracis, protective antigen,spoOA, vaccine, Congo red.

ISSN:0008-4166    

DOI:10.1139/w98-108

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

A batch agitation technique was designed to specify the different parameters that influence the inactivation and adsorption mechanisms of viruses in water. The advantage of this method over the classical procedures is that the kinetic reactions of the different subfractions of the virus population can be described simultaneously. A first set of experiments with phage T7 showed that this phage is rapidly inactivated in a constantly agitated liquid medium. This inactivation rate is highly influenced by temperature, but variation of the pH (from 5 to 9) and increase in salt concentration have no effect on it. The addition of colloidal clay particles (CCPs) of montmorillonite and attapulgite into the liquid medium considerably modifies this behavior, even at very low concentrations (0.025 mg/mL). The experiments show that the viruses react quickly with the particles and that bonding is not permanent. Viruses establish a dynamic equilibrium, which is strongly dependent on physicochemical parameters such as pH, ionic concentrations, and the presence of proteins or protein hydrolysates. A major environmental consequence is that the presence of CCPs seems to effectively protect the coliphage T7 from rapid inactivation.Key words: bacteriophage T7, viruses, inactivation and adsorption kinetics, colloidal particles, protective effect.

ISSN:0008-4166    

DOI:10.1139/w98-117

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

Organic solvent-tolerant mutants ofPseudomonas aeruginosaselected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical ofnalBmultidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems inP. aeruginosademonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis ofmexR, the repressor gene ofmexAB-oprMefflux operon, identified a nonsense mutation and a point mutation in themexRgenes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.Key words:Pseudomonas aeruginosa, organic solvent tolerance, multidrug resistance, MexAB-OprM efflux pump,mexRgene.

ISSN:0008-4166    

DOI:10.1139/w98-127

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form ofCampylobacter upsaliensiscould not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purifiedEscherichia colipeptidoglycan or whole cells ofMicrococcus luteus(Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that containedEscherichia colipeptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells ofM.luteus,only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containingEscherichia colipeptidoglycan. This common autolysin was isolated by adsorbing it fromCampylobacter upsaliensissoluble fractions ontoM.luteuscells and then subjecting these cells to renaturing SDS-PAGE in gels containingEscherichia colipeptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to theM.luteuscells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 ofCampylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin,Campylobacter upsaliensis, zymogram, murein hydrolase.

ISSN:0008-4166    

DOI:10.1139/w98-125

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

Analysis ofSaccharomyces cerevisiaegenome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified fromS.cerevisiaewas almost equally activated by cAMP and cGMP at 3 × 10-6M concentrations of either nucleotide in the presence of Mg2+ions. Interestingly, if Mn2+ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, whileN6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.Key words: cAMP-dependent protein kinase, cAMP, cGMP, yeast, ribosomal protein S10

ISSN:0008-4166    

DOI:10.1139/w98-214

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

To obtain information on cell wall synthesis and its relationship to morphology, we examined the induction of cell extensions of yeast upon the addition of isoamyl alcohol in osmotically fragile mutants that had mutations in genes related to the cell integrity pathway through activation of the mitogen-activated protein kinase cascade. We found that isoamyl alcohol induces cell extensions inpkc1deletion mutants but not in mutants with mutations in genes positioned downstream or upstream of thePKC1gene. These results suggest that Pkc1p functions not only in the integrity pathway but also in the induction. We characterized the elongated cells; many had two or more nuclei. We found no difference in cell surface structure between round and elongated cells from the results of chitin staining and cell wall extraction. Actin cytoskeleton was organized in elongated cells, as well as round cells. Cytochalasin D (0.08 mg/mL) inhibited the formation of actin cable but did not affect the induction of cell extensions.Key words:Saccharomyces cerevisiae,pkc1, isoamyl alcohol, cell extension.

ISSN:0008-4166    

DOI:10.1139/w98-208

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

Fusaproliferin (FP), a toxic metabolite of the world-wide maize pathogensFusarium proliferatumandFusarium subglutinans, was recently found to be a natural contaminant of maize. Its toxic activity on haematopoietic human cell lines and its teratogenic effects on chicken embryos has been recently proved. Therefore a sensitive, rapid, and inexpensive screening test to detect FP in agricultural commodities is necessary to protect human health. FP-hemiglutarate conjugated to modified bovine serum albumin was synthesized, characterized, and used as an antigen for raising polyclonal antibodies by immunizing rabbits. Indirect and competitive ELISA and immunoblotting analyses were performed to determine antibody specificity towards the mycotoxin. The determination of 10 µg of free FP/mL was achieved using antibodies purified by means of affinity chromatography on a FP-lysine-Sepharose column. This unsatisfactory detection limit is due to high background values; thus, this method is not competitive with traditional UV-HPLC methods.Key words: fusaproliferin, ELISA, mycotoxin, immunoassay, corn,Fusarium.

ISSN:0008-4166    

DOI:10.1139/w98-116

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of elevenLactobacillusbrewing spoilage organisms, including one or more ofL.brevis,L.buchneri, L.casei-alactosus,L.plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases,Lactobacillussurface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection ofLactobacillusbrewing spoilage organisms.Key words: brewing spoilage organism, immunoassay,Lactobacillus, monoclonal antibodies, surface antigens.

ISSN:0008-4166    

DOI:10.1139/w98-220

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

We examined changes in mouse fecal microflora after various dietary components andBacillus subtilis(natto) spores were delivered by intubation. The administration of intact spores ofBacillus subtilis(natto) did not affect fecalEnterobacteriaceaeandEnterococcusspp. in all three diet groups; on the other hand, it did affect fecalBacteroidaceaeandLactobacillusspp., depending upon the diets fed. The administration of autoclaved spores did not alter fecal microflora. In vitro cultures ofLactobacillus murinusobtained from mouse feces, together withBacillus subtilis(natto) under aerobic conditions as a mixed culture, revealed that the growth ofL. murinuswas enhanced by the addition of intact spores ofBacillus subtilis(natto). This enhancement of growth was displayed only in media containing either sucrose, glucose, maltose, or fructose but not in media containing cornstarch, soluble starch, or microcrystalline cellulose. From these results it was evident that some metabolites ofBacillus subtilis(natto) produced during germination and (or) outgrowth of spores of this strain, requiring monosaccharides or oligosaccharides, participated in the enhancement of the growth ofL. murinus.Key words:Bacillus subtilis(natto),Lactobacillus, probiotics, intestinal microflora, dietary components.

ISSN:0008-4166    

DOI:10.1139/w98-206

出版商:NRC Research Press    

年代:1999

数据来源: NRC

摘要:

Typing and identification of 60 marine psychrophilic and psychrotrophic vibrios isolated from the north-western Pacific Ocean and coastal environment of Japan were performed by restriction fragment length polymorphism analysis on the basis of polymerase chain reaction amplified 16S rDNA. We obtained 15 operational taxonomic units (OTUs) by digestion with four restriction endonucleases (HhaI,DdeI,RsaI, andSau3AI); four large groups were obtained from the neighbor-joining method. Significant differences were observed in OTU composition between isolates from the deep sea and coastal areas.Vibrio marinusandPhotobacteriumspecies were the dominant culturable vibrios in the deep sea areas, whileVibriosplendiduslike species were the dominant culturable vibrios in a coastal area of Japan.Key words: restriction analysis,Vibrio,Photobacterium, Vibrio marinus(Moritella marina),Vibrio splendidus.

ISSN:0008-4166    

DOI:10.1139/w98-128

出版商:NRC Research Press    

年代:1999

数据来源: NRC