bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oriented Adhesion of
Escherichia coli
to Polystyrene Particles
Authors: Jones Joseph F. , Feick Jason D. , Imoudu Daniel , Chukwumah Nkiru , Vigeant Margot , Velegol Darrell 
Published: November 2003

ABSTRACT

The adhesion of nonflagellated
Escherichia coli
strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.
Gene Dosage Effect of
l
-Proline Biosynthetic Enzymes on
l
-Proline Accumulation and Freeze Tolerance in
Saccharomyces cerevisiae
Authors: Terao Yukiyasu , Nakamori Shigeru , Takagi Hiroshi 
Published: November 2003

ABSTRACT

We have previously reported that
l
-proline has cryoprotective activity in
Saccharomyces cerevisiae
. A freeze-tolerant mutant with
l
-proline accumulation was recently shown to carry an allele of the
PRO1
gene encoding γ-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of
l
-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant γ-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three
l
-proline biosynthetic enzymes, including Δ
1
-pyrroline-5-carboxylate reductase, which converts Δ
1
-pyrroline-5-carboxylate into
l
-proline, on
l
-proline accumulation and freeze tolerance in a non-
l
-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on
l
-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant γ-glutamyl kinase and the wild-type γ-glutamyl phosphate reductase was effective for
l
-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Δ
1
-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of
l
-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular
l
-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.
Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses
Authors: Lamothe Gilbert Thierry , Putallaz Thierry , Joosten Han , Marugg Joey D. 
Published: November 2003

ABSTRACT
A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.
Members of a Readily Enriched β-Proteobacterial Clade Are Common in Surface Waters of a Humic Lake
Authors: Burkert Ulrike , Warnecke Falk , Babenzien Dieter , Zwirnmann Elke , Pernthaler Jakob 
Published: November 2003

ABSTRACT

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. β-Proteobacteria from a cosmopolitan freshwater lineage related to
Polynucleobacter necessarius
(beta II) were rapidly enriched in all treatments. In contrast, members of the class
Actinobacteria
did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment.
Actinobacteria
represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.
Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of
Escherichia coli
Authors: Overhage Jörg , Steinbüchel Alexander , Priefert Horst 
Published: November 2003

ABSTRACT

The
vaoA
gene from
Penicillium simplicissimum
CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in
Escherichia coli
XL1-Blue under the control of the
lac
promoter, together with the genes
calA
and
calB
, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of
Pseudomonas
sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain
E. coli
XL1-Blue(pSK
vao
mP
calA
m
calB
) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter
−1
. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter
−1
after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation,
E. coli
XL1-Blue(pSK
vao
mP
calA
m
calB
) was used to produce ferulic acid from eugenol and, subsequently,
E. coli
(pSK
ech
E/H
fcs
) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter
−1
, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter
−1
. The genes
ehyAB
, encoding eugenol hydroxylase of
Pseudomonas
sp. strain HR199, and
azu
, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in
E. coli
XL1-Blue.
Characterization of aLactococcus lactisStrain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice
Authors: Chatel Jean-Marc , Nouaille Sebastien , Adel-Patient Karine , Le Loir Yves , Boe Herman , Gruss Alexandra , Wal Jean-Michel , Langella Philippe 
Published: November 2003

ABSTRACTBovine β-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacteriumLactococcus lactisand the characterization of its immunogenicity in mice. We constructed a recombinant strain ofL. lactiscapable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinantL. lactisresulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producingL. lactisstrain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producingL. lactisstrain.
Sampling Natural Viral Communities from Soil for Culture-Independent Analyses
Authors: Williamson Kurt E. , Wommack K. Eric , Radosevich Mark 
Published: November 2003

ABSTRACT

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 × 10
8
g of dry soil
−1
), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5×10
8
g of dry soil
−1
) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.
Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass
Authors: Buchan Alison , Newell Steven Y. , Butler Melissa , Biers Erin J. , Hollibaugh James T. , Moran Mary Ann 
Published: November 2003

ABSTRACT

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of
Spartina alterniflora
detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α-
Proteobacteria
and one with the
Cytophagales
) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α-
Proteobacteria
and two ascomycete fungi (
Phaeosphaeria spartinicola
and environmental isolate “4clt”).
Plant Transformation by Coinoculation with a Disarmed
Agrobacterium tumefaciens
Strain and an
Escherichia coli
Strain Carrying Mobilizable Transgenes
Authors: Pappas Katherine M. , Winans Stephen C. 
Published: November 2003

ABSTRACT

Transformation of
Nicotiana tabacum
leaf explants was attempted with
Escherichia coli
as a DNA donor either alone or in combination with
Agrobacterium tumefaciens
. We constructed
E. coli
donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an
E. coli
strain expressing the IncN-type conjugation system was coinoculated with a disarmed
A. tumefaciens
strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from
E. coli
to the disarmed
A. tumefaciens
strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct
A. tumefaciens
strains containing binary vectors and could simplify the creation of transgenic plants.
Detection and Identification of
Lactobacillus
Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis
Authors: Guan Le Luo , Hagen Karen E. , Tannock Gerald W. , Korver Doug R. , Fasenko Gaylene M. , Allison Gwen E. 
Published: November 2003

ABSTRACT

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10
8
to 10
9
CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the
Lactobacillus acidophilus
group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the
L. acidophilus
group.
Hae
III-ARDRA was used for preliminary identification of isolates in the
L. acidophilus
group and to identify
Lactobacillus reuteri
and
Lactobacillus salivarius. Mse
I-ARDRA generated unique patterns for all species of the
L. acidophilus
group, identifying
Lactobacillus crispatus
,
Lactobacillus johnsonii
, and
Lactobacillus gallinarum
among crop isolates. The results of our study provide comprehensive knowledge of the
Lactobacillus
microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.
Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi
Authors: van Aarle Ingrid M. , Olsson Pål Axel 
Published: November 2003

ABSTRACT

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi
Scutellospora calospora
and
Glomus intraradices
when colonizing
Plantago lanceolata
. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of
G. intraradices
increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus.
S. calospora
does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for
G. intraradices. G. intraradices
allocated more of its lipids to storage than did
S. calospora
. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.
Ecology of Uncultivated
Oscillospira
Species in the Rumen of Cattle, Sheep, and Reindeer as Assessed by Microscopy and Molecular Approaches
Authors: Mackie Roderick I. , Aminov Rustam I. , Hu Wenping , Klieve Athol V. , Ouwerkerk Diane , Sundset Monica A. , Kamagata Yoichi 
Published: November 2003

ABSTRACT

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium
Oscillospira
spp. was studied.
Oscillospira-
specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the
Oscillospira
clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an
Oscillospira-
specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large
Oscillospira
morphotypes, the presence of
Oscillospira
spp. was still detected by
Oscillospira-
specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of
Oscillospira
species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.
Methodology of Protistan Discovery: from rRNA Detection to Quality Scanning Electron Microscope Images
Authors: Stoeck Thorsten , Fowle William H. , Epstein Slava S. 
Published: November 2003

ABSTRACT
Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences.
Development and Application of a
dapB
-Based In Vivo Expression Technology System To Study Colonization of Rice by the Endophytic Nitrogen-Fixing Bacterium
Pseudomonas stutzeri
A15
Authors: Rediers Hans , Bonnecarrère Victoria , Rainey Paul B. , Hamonts Kelly , Vanderleyden Jos , De Mot René 
Published: November 2003

ABSTRACT

Pseudomonas stutzeri
A15 is a nitrogen-fixing bacterium isolated from paddy rice. Strain A15 is able to colonize and infect rice roots. This strain may provide rice plants with fixed nitrogen and hence promote plant growth. In this article, we describe the use of
dapB
-based in vivo expression technology to identify
P. stutzeri
A15 genes that are specifically induced during colonization and infection (
cii
). We focused on the identification of
P. stutzeri
A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium. Several transcriptional fusions induced in the rice rhizosphere were isolated. Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins.