bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe
Authors: Oehl Fritz , Sieverding Ewald , Ineichen Kurt , Mäder Paul , Boller Thomas , Wiemken Andres 
Published: May 2003

ABSTRACT

The impact of land use intensity on the diversity of arbuscular mycorrhizal fungi (AMF) was investigated at eight sites in the “three-country corner” of France, Germany, and Switzerland. Three sites were low-input, species-rich grasslands. Two sites represented low- to moderate-input farming with a 7-year crop rotation, and three sites represented high-input continuous maize monocropping. Representative soil samples were taken, and the AMF spores present were morphologically identified and counted. The same soil samples also served as inocula for “AMF trap cultures” with
Plantago lanceolata
,
Trifolium pratense
, and
Lolium perenne
. These trap cultures were established in pots in a greenhouse, and AMF root colonization and spore formation were monitored over 8 months. For the field samples, the numbers of AMF spores and species were highest in the grasslands, lower in the low- and moderate-input arable lands, and lowest in the lands with intensive continuous maize monocropping. Some AMF species occurred at all sites (“generalists”); most of them were prevalent in the intensively managed arable lands. Many other species, particularly those forming sporocarps, appeared to be specialists for grasslands. Only a few species were specialized on the arable lands with crop rotation, and only one species was restricted to the high-input maize sites. In the trap culture experiment, the rate of root colonization by AMF was highest with inocula from the permanent grasslands and lowest with those from the high-input monocropping sites. In contrast, AMF spore formation was slowest with the former inocula and fastest with the latter inocula. In conclusion, the increased land use intensity was correlated with a decrease in AMF species richness and with a preferential selection of species that colonized roots slowly but formed spores rapidly.
Association of
Borrelia garinii
and
B. valaisiana
with Songbirds in Slovakia
Authors: Hanincová Klára , Taragelová Veronika , Koci Juraj , Schäfer Stefanie M. , Hails Rosie , Ullmann Amy J. , Piesman Joseph , Labuda Milan , Kurtenbach Klaus 
Published: May 2003

ABSTRACT

In Europe, 6 of the 11 genospecies of
Borrelia burgdorferi
sensu lato are prevalent in questing
Ixodes ricinus
ticks. In most parts of Central Europe,
B. afzelii
,
B. garinii
, and
B. valaisiana
are the most frequent species, whereas
B. burgdorferi
sensu stricto,
B. bissettii
, and
B. lusitaniae
are rare. Previously, it has been shown that
B. afzelii
is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of
B. garinii
and
B. valaisiana
in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing
I. ricinus
ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as
B. garinii
and
B. valaisiana
, whereas questing ticks were infected with
B. afzelii
,
B. garinii
, and
B. valaisiana
. Furthermore, identical
ospA
alleles of
B. garinii
were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of
B. garinii
and
B. valaisiana
but not of
B. afzelii
. This and previous studies confirm that
B. burgdorferi
sensu lato is host associated and that this bacterial species complex contains different ecotypes.
Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in
Clostridium acetobutylicum
ATCC 824
Authors: Zhao Yinsuo , Hindorff Lucia A. , Chuang Amy , Monroe-Augustus Melanie , Lyristis Michael , Harrison Mary L. , Rudolph Frederick B. , Bennett George N. 
Published: May 2003

ABSTRACT

The cyclopropane fatty acid synthase gene (
cfa
) of
Clostridium acetobutylicum
ATCC 824 was cloned and overexpressed under the control of the clostridial
ptb
promoter. The function of the
cfa
gene was confirmed by complementation of an
Escherichia coli cfa
-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing
cfa
were introduced into
C. acetobutylicum
hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the
cfa
gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the
cfa
-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of
cfa
results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a
marR
homologous gene preceding the
cfa
gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.
Recovery and Enumeration of
Cryptosporidium parvum
from Animal Fecal Matrices
Authors: Davies Cheryl M. , Kaucner Christine , Deere Daniel , Ashbolt Nicholas J. 
Published: May 2003

ABSTRACT

Accurate quantification of
Cryptosporidium parvum
oocysts in animal fecal deposits on land is an essential starting point for estimating watershed
C. parvum
loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 10
2
oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces
−1
must be present to be detected.
Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays
Authors: Urakawa Hidetoshi , El Fantroussi Said , Smidt Hauke , Smoot James C. , Tribou Erik H. , Kelly John J. , Noble Peter A. , Stahl David A. 
Published: May 2003

ABSTRACT

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of
Staphylococcus epidermidis
(38 nucleotides) and
Nitrosomonas eutropha
(39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.
A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria
Authors: Holm Claus , Jespersen Lene 
Published: May 2003

ABSTRACT

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with
Staphylococcus aureus
and
Escherichia coli
. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.
In Vivo Tracking ofCampylobacter jejuniby Using a Novel Recombinant Expressing Green Fluorescent Protein
Authors: Mixter Philip F. , Klena John D. , Flom Gary A. , Siegesmund Amy M. , Konkel Michael E. 
Published: May 2003

ABSTRACTCampylobacter jejuniis a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study ofC. jejuniin a variety of niches.C. jejunitransformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally withC. jejuniharboring thegfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined thatC. jejunisynthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+Gr-1+lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+Gr-1−lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b−CD45R+B lymphocytes from the lavage of theC. jejuni-injected mice were associated with green-fluorescentC. jejuni(proportions ranged from 0.75 to 0.77%). Cell-freeC. jejuniwas recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of theC. jejuniF38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate thatC. jejuniassociates with marker-defined cellular subsets in vivo with a novelgfpreporter system and thatC. jejunigenotypic variants can be isolated from both in vitro and in vivo systems.
Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods
Authors: Thimm Torsten , Tebbe Christoph C. 
Published: May 2003

ABSTRACT

A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of
Folsomia candida
(Collembola) and the detection of bacteria in both the gut and tissue.
Specific Detection of
Dehalococcoides
Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes
Authors: Yang Yanru , Zeyer Josef 
Published: May 2003

ABSTRACT

Dehalococcoides ethenogenes
is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of
Dehalococcoides
species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for
Dehalococcoides
species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except
D. ethenogenes
and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of
Dehalococcoides
species. In situ hybridization of probe Dhe1259t with
D. ethenogenes
strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active
Dehalococcoides
species in these enrichment samples.
RecombinantSaccharomyces cerevisiaeExpressing P450 in Artificial Digestive Systems: a Model for Biodetoxication in the Human Digestive Environment
Authors: Blanquet S. , Meunier J. P. , Minekus M. , Marol-Bonnin S. , Alric M. 
Published: May 2003

ABSTRACTThe use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model ofSaccharomyces cerevisiaeexpressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% ± 10.1% [n= 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% ± 2.7% [n= 3]). For the first time, the ability of recombinantS. cerevisiaeto carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% ± 5.8% (n= 3) of the ingestedtrans-cinnamic acid was converted intop-coumaric acid after 4 h of digestion, as well as 8.9% ± 1.6% (n= 3) in the stomach, 13.8% ± 3.3% (n= 3) in the duodenum, 11.8% ± 3.4% (n= 3) in the jejunum, and 6.5% ± 1.0% (n= 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest thatS. cerevisiaemay afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.
Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68°C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation
Authors: Ferris Mike J. , Kühl Michael , Wieland Andrea , Ward David M. 
Published: May 2003

ABSTRACTWe examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68°C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O2and oxygenic photosynthesis demonstrated the existence of physiologically distinctSynechococcuspopulations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation inSynechococcus16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths.
In Situ Accessibility of
Saccharomyces cerevisiae
26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains
Authors: Inácio João , Behrens Sebastian , Fuchs Bernhard M. , Fonseca Álvaro , Spencer-Martins Isabel , Amann Rudolf 
Published: May 2003

ABSTRACT

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of
Saccharomyces cerevisiae
PYCC 4455
T
. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.
Isolation and Characterization of Novel Psychrophilic, Neutrophilic, Fe-Oxidizing, Chemolithoautotrophic α- and γ-
Proteobacteria
from the Deep Sea
Authors: Edwards K. J. , Rogers D. R. , Wirsen C. O. , McCollom T. M. 
Published: May 2003

ABSTRACT

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS
2
), basalt glass (∼10 wt% FeO), and siderite (FeCO
3
), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-
Proteobacteria
most closely related to the heterotrophic bacterium
Marinobacter aquaeolei
(87 to 94% sequence similarity). A second group of isolates are α-
Proteobacteria
most closely related to the deep-sea heterotrophic bacterium
Hyphomonas jannaschiana
(81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.
Development of a Plating Medium for Selection of
Helicobacter pylori
from Water Samples
Authors: Degnan A. J. , Sonzogni W. C. , Standridge J. H. 
Published: May 2003

ABSTRACT

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of
Helicobacter pylori
. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (
Acinetobacter
,
Aeromonas
,
Bacillus
,
Escherichia coli
,
Enterobacter
,
Enterococcus
,
Helicobacter pylori
, and
Pseudomonas
strains) were individually plated onto each of these media. Three organisms (
Acinetobacter
,
E. coli
, and
H. pylori
) were able to grow on all five media. This growth was unacceptable since
Helicobacter
grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (
Flavobacterium
,
Serratia
,
Citrobacter
,
Pasteurella
,
Ochrobactrum
,
Rahnella
, and unidentified molds) and were further adulterated with the eight strains listed above (10
6
CFU of each strain per 100 ml) were spiked with
H. pylori
and were plated. In spite of the heavy mixed microbial load, only
H. pylori
colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of
H. pylori
colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for
H. pylori
from mixed microbial populations in water and reduces the time required to complete the assay.