摘要:

A gnotobiotic bioassay, using potato plantlets derived from single-node explants and grown in test tubes containing potato nodal cutting medium (PNCM), was found to be highly useful for investigations of direct growth promotion by a nonfluorescentPseudomonassp. strain PsJN. Strain PsJN was used to optimize and evaluate this bioassay for purposes of screening other rhizosphere bacteria and identification of Tn5mutants of strain PsJN deficient in growth-promoting properties. The selection of potato cultivar used in this bioassay was critical, as growth promotion of potatoes by strain PsJN was cultivar specific. Inoculated plantlets of cultivars Norchip, Kennebec, Shepody, and Chaleur showed, in root dry weight, a five- to eight-fold increase, two- to three-fold increase, no response, and a decrease of 50%, respectively. Haulm dry weight followed similar trends but was not as consistent an indicator of growth promotion. Bioassay results were not altered to any extent by minor changes in PNCM composition or by slight changes in temperature and light conditions. A rapid method for preparation of bacterial suspensions and inoculation of explants was developed. Inoculation of three explants taken from 6-week-old stock plantlets of cv. Kennebec for each Tn5transconjugate of strain PsJN (total of 1500 transconjugates) enabled the elimination of 93% of those isolates that retained growth-promoting activity. The remaining 7% of isolates were retested and seven were confirmed to have lost growth-promoting ability. Bacteria from different genera were also screened with this bioassay. None of these bacteria increased the growth of potato plantlets, but several inhibited root and haulm growth.Key words: plant growth-promoting rhizobacteria, gnotobiotic, tissue culture, nonfluorescent pseudomonad, bacterium, potato.

ISSN:0008-4166    

DOI:10.1139/m97-117

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Pseudomonas putidaGR12-2R3 promotes the emergence and growth of diverse plant species. Analyses of TnphoAinsertion mutations are revealing bacterial characteristics pertinent to the plant–microbe interaction.Pseudomonas putidaPG269 is a TnphoAinsertion derivative of GR12-2R3 that expresses canola seed exudate-inducible alkaline phosphatase (PhoA) activity. It promoted the growth of canola roots, as well as strain GR12-2R3, and outgrew its parent when they were cocultured in the presence of canola roots or in liquid seed exudate medium. (In contrast, mutant PG126 failed to promote canola root growth and was outgrown by its parent strain.) The PhoA activity of strain PG269 was induced by glucosamine and other sugars; glucosamine inhibited the growth of strain GR12-2R3 and stimulated the growth of strain PG269. Strain PG269 contained two TnphoAinsertions:seiA1:: TnphoAandseiB1:: TnphoA. Strain PG312, which contained only insertionseiA1:: TnphoA, shared all aspects of the PG269 phenotype, except the ability to outcompete strain GR12-2R3 during coculture. InsertionseiA1::TnphoAinterrupted an open reading frame related in sequence to members of the MalF family of sugar transporter subunits. The PhoA-inducing fraction of canola seed exudate was hydrophilic, low in molecular weight, and heat stable. It cochromatographed with basic amino acids and amino sugars, and was inactivated by strains GR12-2R3 and PG269. GeneseiAmay encode a subunit of an ABC transporter with broad specificity for glucose and related sugars whose expression can be induced by exudate sugars.Key words:Pseudomonas putida, canola, exudate, sugar transport, rhizobacterium.

ISSN:0008-4166    

DOI:10.1139/m97-118

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Previous experiments using expression plasmids which overproduce the β and β′ subunits ofEscherichia coliRNA polymerase suggested that regions considerably upstream of the start of therpoBgene, which encodes the β subunit, are required for its efficient synthesis. To further delineate the required regions, a collection of genetic constructs that contained varying amounts of the region either upstream or downstream of the translational start ofrpoBwas assembled. Measurements of β and β′ synthesis andrpoBmRNA production from a series ofrpoBCexpression plasmids indicated that sequences extending more than 43 bp but less than 79 bp upstream ofrpoBare required for the efficient translation ofrpoBmRNA. This result was confirmed by β-galactosidase measurements from a series ofrpoB-lacZfusions that have the same set of end points upstream ofrpoBas the expression plasmids. A second set of gene fusions containing differing amounts of the sequence distal to the start ofrpoBfused in frame tolacZrevealed that more than 29 bp but less than 70 bp ofrpoBwas required for efficient translation.Key words: RNA polymerase,E.coli, translational regulation.

ISSN:0008-4166    

DOI:10.1139/m97-119

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

The fate of a bacterium and two yeast species genetically engineered by insertion of a nucleotide sequence encoding for aprotinin was studied in three different soils.Corynebacterium glutamicumcarried the recombinant gene on plasmid pUN1,Saccharomyces cerevisiaecarried the gene on plasmid p707, and inPichia angusta(formerlyHansenula polymoropha) LR9-Apr8, the gene was chromosomally inserted with eight tandem repeats.Corynebacterium glutamicumpersisted longer than both yeast strains. In a sandy loam of pH 5.9, recovery rates of cultured cells were lower than in a clay silt or a silty sand, with pH values of 7.1 and 6.7, respectively. Generally, persistence at 10 °C was higher than at 20 °C. An adaptation of the genetically engineered strains resulting in higher soil persistence was not observed for any of the three species tested. Competition experiments between nonengineered and genetically engineered strains in presterilized soils revealed a reduced fitness of the engineered strains. However, a more competitiveC.glutamicumpUN1 evolved after reinoculation of cells, preselected by a preceding competition experiment.Key words: ecological risk assessment, genetic engineering, nondeliberate release, soil inoculation, aprotinin.

ISSN:0008-4166    

DOI:10.1139/m97-120

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Polygalacturonase activity from the phytopathogenic fungusBotrytis cinereawas inhibited in vitro by extracellular polyphosphate fromStreptomycessp. A50, as well as other polyphosphates of biological and chemical origin. The extent of inhibition increased with polyphosphate chain length between 20 and 100 Piresidues. Although the activity of polygalacturonase fromB.cinereaappeared not to depend on the presence of cations, inhibition was partially blocked by divalent cations such as Mg2+or Ca2+. Production of polyphosphate inStreptomycessp. A50 was followed by chemical measurements, as well as by in vivo31P-NMR analysis. During the first 2 days of growth, polyphosphate accumulated within the cells, after which it appeared in the broth as an extracellular product. A maximum concentration of extracellular polyphosphate (1 mM Piequivalent) was reached, corresponding to about 25% of the input Pi. NMR analysis suggested that the intracellular form of polyphosphate exists as a mobile soluble pool. In contrast, the extracellular form of polyphosphate appears to be complexed with cations.Key words: polygalacturonase, polyphosphate,Botrytis cinerea,Streptomyces,31P-NMR.

ISSN:0008-4166    

DOI:10.1139/m97-121

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

The catabolism of selected groundwater pollutants by a combined culture ofMycobacterium vaccaeand aRhodococcussp. (strain R-22) was investigated. TheM.vaccae– R-22 combined culture was five times more effective in mineralizing benzene than either organism alone.Mycobacterium vaccaeoxidized benzene to phenol, and R-22 catabolized the phenol to cellular components and CO2. Benzene did not support growth ofM.vaccae, R-22, or the combined culture. Optimization of ratios of the two species indicated that the maximum mineralization of benzene occurred at an initial ratio of 75%M.vaccaeto 25% R-22. Cell fractionation of the combined culture after mineralization of [U-14C]benzene indicated that 10% of the benzene carbon was incorporated into cell material, and of this 45% was present in protein and 20% in nucleic acids. This suggested that minimally one species could utilize the products of benzene as a nutrient source. TheM.vaccae– R-22 combined culture catabolized ethylbenzene and chlorobenzene without the accumulation of phenolic intermediates, which are inhibitory toM.vaccae'sability to degrade the parent compounds. This study demonstrates that defined mixed cultures may be useful in studying the effects of environmental pollutant degradation on microbial ecosystems and mineralization of these pollutants by the ecosystem.Key words: biodegradation, groundwater pollutant,Mycobacterium vaccae,Rhodococcussp.

ISSN:0008-4166    

DOI:10.1139/m97-122

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Immunofluorescence colony staining (IFC) and a new technique using spiral plating combined with IFC were evaluated for the soft-rot pathogenErwinia carotovorasubsp.atrosepticain witloof chicory. Target bacteria could be detected in platings at various dilutions of plant washings. Brilliance of the stained colonies ofE.carotovorasubsp.atrosepticawas high. Spiral plating, used for both the plating of the bacteria and for the delivery of the conjugated antiserum, had a positive effect on the reduction of the background compared with the staining of the bacteria. The combination of spiral plating and IFC proved to be a functional tool for the quantification of target and nontarget bacteria and the isolation of target bacteria as pure culture from IFC-positive colonies. The method uses less conjugated antiserum than traditional IFC and produces results with very small variation within replications. The recovery of the bacteria in both pure culture and plant washing is significantly higher than the recovery using crystal violet pectate medium.Key words: soft rot, witloof chicory, detection,Erwinia carotovorasubsp.atroseptica.

ISSN:0008-4166    

DOI:10.1139/m97-123

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Symbiotic bacteria associated with theMedicagogenus are separated into two closely related species namedSinorhizobium melilotiandSinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiateS.melilotifromS.medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species ofRhizobiumandSinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific forS.medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs ofS.meliloti,Sinorhizobium fredii, andSinorhizobium sahelibut not with the DNA ofS.medicae. SinceS.saheliandS.frediido not nodulateMedicago, 16Smed and 16Smfs can be routinely used to characterize the twoSinorhizobiumspecies nodulatingMedicagofrom pure cultures or fromMedicagoroot nodules. Fifty strains isolated from eight annualMedicagospecies were then characterized by using colony hybridizations.Sinorhizobium melilotiwas more frequently obtained (>80% isolates) than wasS.medicae. BothSinorhizobiumspecies seemed to be trapped by annualMedicagoand no plant-host specificity was detected.Key words:Sinorhizobium meliloti,Sinorhizobium medicae,Medicago, oligonucleotide probe, 16S rDNA gene.

ISSN:0008-4166    

DOI:10.1139/m97-124

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Xanthomonas oryzaepv.oryzaeis the causal agent of bacterial leaf blight, a serious disease of rice. Spontaneous loss of virulence inX.oryzaepv.oryzaeis associated with mutants that produce reduced levels of extracellular polysaccharide. Evidence is presented that these mutants accumulate in long-term stationary phase cultures but are not obtained from exponentially growing cultures. A number of independentspv(stationary-phase variation) mutants have been isolated and characterized. These mutants differ from each other with respect to the stability of the mutant phenotype, extent of loss of virulence, ability to outcompete wild type cells in stationary-phase cocultures, and hypersensitivity to hydrogen peroxide.Key words: stationary-phase variation, spontaneous loss of virulence,Xanthomonas oryzae.

ISSN:0008-4166    

DOI:10.1139/m97-125

出版商:NRC Research Press    

年代:1997

数据来源: NRC

摘要:

Deleya aesta134 grows optimally at 200 mM Na+in a chemically defined medium but at 10 mM Na+only after an extended lag period which was reduced if the cells that grew were reinoculated into medium of the same low Na+concentration. Cells that eventually grew at low Na+formed colonies on agar containing 17 mM Na+in the agar supernatant (the liquid released when the agar was compacted). Cells of the parent failed to form colonies at this Na+concentration when 102cells were plated. Colonies that formed on low Na+agar differed in appearance from colonies of the parent and three colony types were distinguished. When 106cells ofD.aestagrown in liquid medium containing optimum Na+were spread on plates containing 17 mM Na+, a few variant colonies first appeared on day 4 and then increased in numbers over a 20-day period. In nine similar cultures the yield of colonies varied over a 3-log range. Fluctuation tests applied to the numbers arising from the similar cultures after different periods of incubation of the plates showed that the ratio of the variance to the mean was much greater than one initially and then increased with time. A total of seven different variants were isolated. These could be distinguished by the colony type formed, the length of the lag time preceding the first appearance of colonies, and the rate of colony accumulation on low (and in one case, high) Na+plates. The variants retained their distinctive characteristics when replated at low Na+after growth at optimum Na+. Differences in lag time and rate of colony accumulation were related to differences in Na+requirement of the variants and to the presence of other colonies on the plates. The variants appear to arise as the result of random mutations in the growing culture. There was no evidence of adaptive mutation.Key words:Deleya aesta, marine bacteria, variants, Na+response, colony accumulation, adaptive mutation.

ISSN:0008-4166    

DOI:10.1139/m97-126

出版商:NRC Research Press    

年代:1997

数据来源: NRC