摘要:

An effective method is described for selectively isolating the actinomycete genusActinobispora.Gellan gum plus calcium chloride significantly stimulated aerial mycelium formation ofActinobispora yunnanensisIFO 15681 so that this genus was readily recognized on the isolation plate. A new medium, HVG, containing calcium chloride and gellan gum as a solidifying agent was thus developed based on humic acid - vitamin agar. A number ofActinobisporastrains were successfully isolated on this medium from 14 soil samples, which were collected in Canada, France, Japan, the United Kingdom, and the United States. To our knowledge, this is the first isolation ofActinobisporastrains from locations other than China.Key words:Actinobispora, gellan gum, selective isolation, actinomycete.

ISSN:0008-4166    

DOI:10.1139/w97-117

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The biosynthesis and role of fructose 2,6-bisphosphate (Fru-2,6-P2) in carbohydrate metabolism during induction of an amylolytic system in Aspergillus oryzae was studied. Fluctuations in Fru-2,6-P2were not dependent on the external glucose concentration during induction, whereas the level of Fru-2,6-P2increased significantly when the oxygen concentration was diminished. Phosphofructokinase II (PFK II) of A. oryzae was sensitive to phosphorylation in vitro by the catalytic subunit of cyclic AMP dependent protein kinase, which increased the Vmax(twofold), although the Km(0.7 mM) remained unchanged. Phosphofructokinase I was neither activated by micromolar Fru-2,6-P2nor inhibited by high ATP concentrations. The activity of fructose-1,6-bisphosphatase (FBPase) was subject to strong inhibition by Fru-2,6-P2. Addition of glucose to cultures under gluconeogenic conditions caused a decrease of approximately 40% in the FBPase activity within 4 min. These results indicate that the effect of Fru-2,6-P2in A. oryzae could preferentially control gluconeogenesis. The addition of 0.1 M glucose under gluconeogenic culture conditions also showed that Fru-2,6-P2fluctuations appeared to be, at least in short term, more closely related to temporal changes in the hexose-6-phosphate concentration.Key words:Aspergillus oryzae; fructose-2,6-bisphosphate; phosphofructokinase II (PFK II); cyclic AMP; gluconeogenesis control.

ISSN:0008-4166    

DOI:10.1139/w97-129

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The occurrence ofAcetobacter diazotrophicuswas directly demonstrated in plant tissues using a species-specific oligonucleotide probe and polymerase chain reaction (PCR) amplification of a 411-bp product. The oligonucleotide probe was derived from the sequence of a highly variable region of 23S rDNA and its specificity was tested with membrane-bound nucleic acids of 112 different microorganisms in hybridization experiments. It was found to be able to discriminateAcetobacter diazotrophicusfrom otherAcetobacterspp. and other reference organisms. PCR amplification from pure cultured cells or colonies showed that the method was sensitive enough to detect as few as 200 cells in the reaction. The presence ofAcetobacter diazotrophicusin tissues of micropropagated sugarcane plants inoculated with either this bacterium or a mixture of this bacterium andHerbaspirillum seropedicaewas demonstrated by PCR amplification.Acetobacter diazotrophicuscould also be detected by the PCR method in field-grown sugarcane plants, as well as in certain cultivars ofPennisetum purpureumSchumach but not in\i maize, sweet potato, and two samples of weed plants grown within or outside of a sugarcane field.The addition of 1% polyvinylpolypyrrolidone during preparation of the field samples, especially with root tissues, improved the amplificability of the target sequence. The minimum level of detection of this bacterium in sugarcane tissue using the universal 1440 and AD species-specific primers was about 105bacterial cells/g of fresh plant material. The sensitivity could be improved 10-fold by probing immobilized PCR products containing the target region with the32P-labeled oligonucleotide AD.Key words:Acetobacter diazotrophicus, diazotrophic endophytes, specific rRNA-targeting oligonucleotides, polymerase chain reaction (PCR).

ISSN:0008-4166    

DOI:10.1139/w97-116

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

We previously demonstrated the existence of bacteria degrading carboxy-methyl-cellulose in the gut of the phytophagous snailHelix aspersaand foundEnterobacteriaceaepredominating in the intestine of snails dissected in aerobic conditions. Here, we investigated the effects of several nutritional treatments on the snail's microflora. Food sterilization led to increased snail growth and reduced cellulase activity in the crop, suggesting a noxious effect of microbial exogenous cellulases. A second aim of this study was to look for anaerobic bacteria. No strict anaerobic cellulolytic, homoacetogenic, or methanogenic bacteria were enriched from the gut. However, a motile Gram-positive homolactic coccobacillus, grown in anaerobic conditions, dominated in the snail's intestine (1.57 x 109± 0.10 x 109cells.g-1intestine). It was identified asEnterococcus casseliflavus. Its occurrence in the intestine ofH. aspersais discussed with regard to the snail's digestive processes and the presence of a fecal mucous ribbon. A possible snail-bacterium synergistic action is suggested.Key words: snail,Helix aspersa, gut,Enterococcus casseliflavus, fermentative homolactic bacterium, antibiotics.

ISSN:0008-4166    

DOI:10.1139/w97-120

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

We have examined verotoxin (VT) binding to cell surface proteins. When Vero or globotriaosylceramide (Gb3) deficient Vero (VRP) cells were incubated with125I-labelled verotoxin 2 (VT2) and disuccinimidyl suberate cross-linker, SDS-PAGE of cell lysates showed radiolabelled bands at 44, 50, 60, 86, 102, and 138 kDa. When125I-labelled verotoxin 1 (VT1) was cross-linked, radioactive bands occurred at 51, 67, 101, 160, 188, and 232 kDa. In contrast,125I-labelled VT1 B subunit produced a single radioactive band migrating at 50 kDa. CHO cells did not bind labelled VT. VT2 binding to VRP cells fit a rectangular hyperbola suggesting a single class of binding sites. In contrast, VT1 and VT1 B subunit binding to VRP cells was best fit by sigmoidal curves suggesting the presence of positive cooperativity between at least two binding sites. Scatchard analysis of VT2 binding data yielded 3.5 times 109molecules bound/ µg of cell protein with an equilibrium dissociation constant (KD) of 13 nM. The apparent KDwas 9.7 nM for VT1 and 73.2 nM for VT1 B subunit. These results indicate that VT binds to a protein, or proteins, on the surface of susceptible cells and that there appear to be differences between VT1 and VT2 binding. Interactions between VT1 or VT2 and the proteins demonstrated here may be important in the biological activity of VT.Key words: verotoxin, protein receptors, hemolytic uremic syndrome,Escherichia coli.

ISSN:0008-4166    

DOI:10.1139/w97-123

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Reverse transcriptase - polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5 prime -carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.Key words: quantitative RT-PCR, poliovirus, sewage, seawater.

ISSN:0008-4166    

DOI:10.1139/w97-128

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

In addition to 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O), biphenyl-grown cells ofComamonas testosteroniB-356 were shown to produce a catechol 2,3-dioxygenase (C2,3O). B1,2O showed strong sequence homology with B1,2Os found in other biphenyl catabolic pathways, while partial sequence analysis of the C2,3O of B-356 suggested a relationship withxylEII-encoded C2,3O. The coexistence of twometa-cleavage dioxygenases in this strain prompted a comparison between the catalytic properties of the two enzymes. C2,3O has a much broader substrate specificity than native or His-tagged B1,2O: both enzymes were inhibited by chlorocatechols, but B1,2O was more sensitive than C2,3O. The results are discussed in terms of the physiological implications of interaction between metabolites from the lower biphenyl-chlorobiphenyl pathway and enzymes of the upper pathway.Key words: chlorobiphenyl, catabolism, dioxygenase, nucleotide sequence, enzyme kinetics.

ISSN:0008-4166    

DOI:10.1139/w97-119

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Bdellovibrio bacteriovorus109J is an obligate intraperiplasmic predator of other Gram-negative bacteria. Collision with a suitable prey cell initiates a developmental sequence ultimately resulting in the destruction of the prey cell and the production of progeny bdellovibrios. Two-dimensional gel analysis of patterns of protein synthesis at various times in a synchronously growing culture ofBdellovibrio bacteriovorus109J revealed over 30 polypeptides whose syntheses are developmentally regulated. The majority of these polypeptides fall into nine categories: attack phase specific or one of eight different kinetic groups expressed during the intraperiplasmic growth phase. The results indicate thatBdellovibrio bacteriovorus109J has a complex system for regulating gene expression during its developmental cycle.Key words: gene regulation, development, two-dimensional gels,Bdellovibrio bacteriovorus.

ISSN:0008-4166    

DOI:10.1139/w97-109

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

SubtypingNeisseria meningitidisby methods that rely on monoclonal antibody (mAb) reactivity results in an unusually high number of strains that are not subtypeable. To subtype 48 strains isolated (1993-1994) in the province of Quebec that were not subtypeable by mAb-based techniques, we used DNA sequencing of the variable regions ofporA,a gene that encodes the class 1 outer membrane protein. We assigned subtypes to all the previously nonserosubtypeable isolates and identified some novel subtypes. Because our sequencing strategy included the promoter region ofporA,different isolates were compared in their sequences of theporApromoter region. A poly(G) stretch lies between the -10 and -35 regions of the promoter; replacement of a G residue by an A residue in this region resulted in loss of expression ofporA. No correlation was found between the number of G residues in the poly(G) stretch and the level of expression; a minimum of 10 G residues is required in this stretch for expression ofporA. One isolate expressed no class 1 outer membrane protein because of the insertion sequence IS1301in the coding region ofporA. Another isolate did not express the protein owing to a frame-shift mutation within the coding region ofporA. Sequencing ofporAallowed assignments of subtypes to previously uncharacterized isolates and provided insights about the regulation of expression of this gene inN. meningitidis.Key words:Neisseria meningitidis, outer membrane proteins, subtyping, PorA, DNA sequencing.

ISSN:0008-4166    

DOI:10.1139/w97-121

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

An antifreeze protein secreted to the growth medium by the plant growth promoting rhizobacteriumPseudomonas putidaGR12-2 was purified to apparent homogeneity. The purified protein has a molecular mass of 164 ± 15 kDa and an isoelectric point of 5.3, contains both carbohydrate and lipid moieties, and is relatively rich in glycine and alanine. The properties of the purified antifreeze protein are similar to the properties previously reported for bacterial ice-nucleation proteins. In fact, the purified antifreeze protein also displays a low level of ice-nucleation activity. Removal of approximately 92 kDa of carbohydrate from the 164-kDa antifreeze glycoprotein did not noticeably alter the antifreeze activity of the molecule, although it did diminish the ice-nucleation activity. This is the first report of an antifreeze protein that also is active as an ice-nucleation protein.Key words: antifreeze protein, plant growth promoting rhizobacteria, freezing tolerance, ice-nucleation protein.

ISSN:0008-4166    

DOI:10.1139/w97-126

出版商:NRC Research Press    

年代:1998

数据来源: NRC