摘要:

In the 7 years since the first publications describing phage-displayed peptide libraries, phage display has been successfully employed in a variety of research. Innovations in vector design and methods to identify target clones account for much of this success. At the same time, not all ventures have been entirely successful and it appears that phage and host biology play important roles in this. A key issue concerns the role played by a displayed peptide or protein in its successful expression and incorporation into virions. While few studies have examined these issues specifically in context of phage display, the literature as a whole provides insight. Accordingly, we review phage biology, relevant aspects of host biology, and phage display applications with the goals of illustrating (i) relevant aspects of the interplay between phage-host biology and successful phage display and (ii) the limitations and considerable potential of this important technology.Key words: bacteriophage M13, phage display, pIII, pVIII, expression libraries.

ISSN:0008-4166    

DOI:10.1139/w98-015

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

ARhodococcusspecies, the best of 99 oil-emulsifying bacteria isolated from globally distributed seawater samples, was characterized. The bacterium produced very stable oil-in-water emulsions from different crude oils with various contents of aliphatic and aromatic compounds by utilizing the C11to C33n-alkanes as carbon and energy sources. The presence of alkanes induced the formation of a hydrophobic cell surface that permitted oil-associated exponential growth and where an extensive emulsification of the residual oil and accumulation of acidic oxidation products occurred. The acidic products were consumed in a second step characterized by linear growth and an increasing number of cells growing in the water phase. Adhesion of cells resulted in some stabilization of oil droplets, but the most extensive emulsification occurred at the end of the exponential phase and coincided with an increasing number of cells in the water phase. No surfactant could be detected in the water phase during exponential growth, but a polymeric compound with emulsifying activity, tightly bound to the oil droplets, could be isolated. This suggests that the emulsification was caused by the release of the hydrophobic cell surface discarded by the cells during conditions of growth limitations.Key words:Rhodococcus,emulsification, adhesion,n-alkanes, hydrophobicity.

ISSN:0008-4166    

DOI:10.1139/w98-005

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Patterns of heat shock gene transcription and translation, as well as trehalose content, were investigated in both glucose (repressed) and acetate (derepressed) grown cells ofSaccharomyces cerevisiaeduring heat shock and subsequent return of cells to 25°C. Heat-shocked cells (37°C for 30 min), grown in either glucose- or acetate-supplemented media, initially acquired high thermotolerance to a 50°C heat stress, which was progressively lost when cultures were allowed to recover at 25°C and subsequently exposed to a second heat stress. In all cases, with the notable exception of repressed cells of a relatively thermosensitive strain, inhibition of protein synthesis and coincident decrease in trehalose accumulation during the heat shock had little effect on the kinetics of loss of thermotolerance. Heat shock at 37°C elicited a marked increase in transcription and translation of genes encoding major heat shock proteins (hsps). During recovery at 25°C, both metabolic activities were suppressed followed by a gradual increase in hsp mRNA transcription to levels observed prior to heat shock. De novo translation of hsp mRNAs, however, was no longer observed during the recovery phase, although immuno- detection analyses demonstrated persistence of high levels of hsps 104, 90, 70, and 60 in cells throughout the 240-min recovery period. In addition, while heat shock induced trehalose was rapidly degraded during recovery in repressed cells, levels remained high in derepressed cells. Results therefore indicated that the progressive loss of induced thermotolerance exhibited by glucose- and acetate-grown cells was not closely correlated with levels of hsp or trehalose. It was concluded that both constitutive and de novo synthesized hsps require heat shock associated activation to confer thermotolerance and this modification is progressively reversed upon release from the heat-shocked state.Key words: thermotolerance, hyperthermic recovery, hsp transcription, hsp translation, trehalose.

ISSN:0008-4166    

DOI:10.1139/w98-006

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.Key words: pBluescript, plasmid, stress, fitness, starvation.

ISSN:0008-4166    

DOI:10.1139/w98-020

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Two hundred and fourteen isolates ofSalmonella typhisubmitted to our laboratory between 1992 and 1996 were tested for susceptibility to 20 antimicrobial agents. Forty-eight of the 214 isolates (22.4%), recovered from individuals who had travelled in South Asia, were multiresistant. Forty-four of the 48 isolates were resistant to ampicillin, chloramphenicol, tetracycline, streptomycin, sulfamethoxazole, trimethoprim, cotrimoxazole, ticarcillin, and piperacillin; the other four isolates were resistant to four to six agents. Forty-two of the multiresistant isolates belonged to Vi phage type E1, two isolates from the Punjab State belonged to phage type A, another from the Punjab State belonged to phage type E3, one isolate from Pakistan belonged to type M1, and one isolate from India belonged to type J1. Plasmids from 45 of 48 isolates showed a temperature-sensitive mechanism of transfer toEscherichia coliK-12 strains, characteristic of HI incompatibility group plasmids. The majority of plasmids had an estimated molecular weight of 120 MDa and encoded both citrate utilization and mercury resistance. Plasmids from three isolates had an estimated molecular weight of 112-115 MDa; one of these isolates encoded citrate utilization but not mercury resistance. Analysis of isolates by pulsed-field gel electrophoresis after digestion withXbaI andSpeI indicated that the majority of multiresistant isolates shared a common restriction profile, while four isolates had unique patterns.Key words: typing, multiresistant,Salmonella typhi.

ISSN:0008-4166    

DOI:10.1139/w98-012

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

A 4.1-kb fragment of chromosomal DNA from the lignocellulose-decomposing actinomyceteStreptomyces viridosporusT7A was previously found to encode a lignin peroxidase gene. However, when cloned intoEscherichia coliin pBSKS+, peroxidase activity was not expressed. When cloned in pIJ702 inStreptomyces lividans, the gene was expressed in a peroxidase positive background, owing to the production byS. lividansof its own extracellular peroxidases. To circumvent these problems, the DNA was cloned into the commercial expression vector pIC9 for extracellular expression in the yeastPichia pastoris. Yeast transformants, however, expressed two activities, extracellular peroxidase and an extracellular endoglucanase. The enzymes were not expressed by the yeast cells alone or by yeast cells with pIC9 without the insert. Expression of the enzymes by only those transformants expressing the 4.1-kb DNA was confirmed by Western blot analyses, by nondenaturing activity gel staining, and by spectrophotometric enzyme assays of extracellular culture filtrates. Activity gel staining showed that the two activities resided in different proteins and the peroxidase expressed was similar to ALip-P3, one of the isoenzymes of lignin peroxidase of theS. viridosporusT7A wildtype. Other evidence indicated that in the transformants, the peroxidase and endoglucanase genes in the 4.1-kb insert were controlled by the methanol-inducible AOX1 yeast promoter in pIC9, since their expression was induced by methanol. In the best transformants, extracellular production of peroxidase by recombinantP. pastoriscultures was significantly higher than typically observed inS. viridosporus. The results also indicate that lignocellulose catabolism genes may be clustered on theS. viridosporuschromosome.Key words: lignocellulose, degradation,Streptomyces, peroxidase, endoglucanase, cloning, pIC9,Pichia pastoris.

ISSN:0008-4166    

DOI:10.1139/w98-010

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The role ofPseudomonas tolaasiias an important pathogen of the common mushroomAgaricus bisporusis difficult to study in the microbially complex growth medium used for mushroom production. Two strains ofP. tolaasiithat had been marked with kanamycin resistance andxylEgenes were introduced individually into casing soil over mushroom compost. Survival studies revealed thatP. tolaasiinumbers in casing soil over mushroom compost decreased a 1000-fold in the first 9 days and then remained relatively stable over the rest of the monitoring period. The presence of the pathogenic colony form and the nonpathogenic colony variant was monitored on mushroom caps and in mushroom compost to detect any phenotypic variation while incubated in these environments. Reversion from the nonpathogenic to pathogenic form was detected following isolation and culture from diseased mushroom caps. Inoculation of the marked strains directly onto the cap or into compost beds seeded withA. bisporusresulted in the appearance of brown blotch symptoms.Key words:Pseudomonas tolaasii,Agaricus bisporus, phenotypic variation, mushroom disease.

ISSN:0008-4166    

DOI:10.1139/w98-003

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

TheBacillus subtilisglutamyl-tRNA synthetase (GluRS), encoded by thegltXgene, aminoacylates its homologous tRNAGluand tRNAGlnwith glutamate. This gene was cloned with its sigmaApromoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 forEscherichia coliandB. subtilis. Transformation ofB. subtiliswith this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation ofE. coliwith this plasmid gave no recombinants, but transformation with plasmids bearing an alteredgltXwas successful. These results indicate that the presence ofB. subtilisglutamyl-tRNA synthetase is lethal forE. coli, probably because this enzyme glutamylates tRNA1Glnin vivo as it does in vitro.Key words: glutamyl-tRNA synthetase overproduction,Bacillus subtilis, toxicity,Escherichia coli.

ISSN:0008-4166    

DOI:10.1139/w98-014

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

An enzyme immunoassay has been developed that confirms identity and estimates cell numbers ofBradyrhizobium japonicumandRhizobium leguminosarumbv.viciaein broth culture. Total testing time required is less than 10 min and no specialized equipment is required. The assay involves a simultaneous and brief reaction of cells, primary antibody, and secondary antibody - enzyme conjugate in aqueous suspension, after which the cells are captured on a 0.45- µm-pore-size syringe filter membrane. Upon addition of enzyme substrate, cell-bound antibody-enzyme conjugate converts the substrate to a blue-green reaction product within the transparent body of the syringe filter. The assay can be quantified by stopping the reaction with acid, extracting the coloured substrate reaction product from the syringe filter, and measuring absorbance using a spectrophotometer. The assay was developed using cultures of rhizobia and bradyrhizobia but should be applicable to liquid cultures of most microorganisms.Key words:Bradyrhizobium, enzyme immunoassay,Rhizobium, syringe filter.

ISSN:0008-4166    

DOI:10.1139/w98-001

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

A two-step broth replacement method was used to induceAzospirillum brasilenseCd bacteria to flocculate in vitro. Nonflocculated and flocculated cells were compared with regard to total cellular lipid composition, fatty acid profiles, and poly-beta-hydroxybutyrate (PHB), protein, and carbohydrate contents. The fatty acid profiles of nonflocculated and flocculated cells were qualitatively identical. Two unsaturated fatty acids, octadecanoate (18:1cis-9) and hexadecanoate (16:1cis-9), accounted for approximately 80% of the total fatty acid content in both phenotypes. The major lipids in nonflocculated and flocculatedA. brasilenseCd cells were phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. The process of flocculation also resulted in the synthesis de novo of a glycolipid and cardiolipin. Flocculation also resulted in a decrease in total cellular protein and lipid content and a proportional increase in total cellular PHB and carbohydrate content. Results indicated that the two-step broth replacement procedure was an effective means for the in vitro production of the stress-tolerantA. brasilenseCd cells with high PHB contents, which are desirable in commercial agricultural inocula. The PHB content of flocculated cells reached 60-65% of cell dry weight.Key words:Azospirillum,flocculation, poly-beta-hydroxybutyrate, PHB, lipid, protein, carbohydrate, fatty acid.

ISSN:0008-4166    

DOI:10.1139/w98-002

出版商:NRC Research Press    

年代:1998

数据来源: NRC