bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phylogenetic Composition of Bacterioplankton Assemblages from the Arctic Ocean
Authors: Bano Nasreen , Hollibaugh James T. 
Published: February 2003

ABSTRACTWe analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domainBacteria: α (36%)-, γ (32%)-, δ (14%)-, and ε (1%)-Proteobacteria;Cytophaga-Flexibacter-Bacteroidesspp. (9%);Verrucomicrobiumspp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 andRoseobactergroups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related toCytophagaandPolaribacterspecies were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) withAlteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) withOceanospirillumsp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to α-Proteobacteria(dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to γ-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.
Displacement of
Escherichia coli
O157:H7 from Rumen Medium Containing Prebiotic Sugars
Authors: de Vaux Albane , Morrison Mark , Hutkins Robert W. 
Published: February 2003

ABSTRACT

A fed-batch, anaerobic culture system was developed to assess the behavior of
Escherichia coli
O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10
10
and 10
4
cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of
E. coli
O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10
6
to 10
9
CFU/ml within 24 h. Most strains of
E. coli
O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude
E. coli
O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter,
E. coli
O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as
l
-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever
E. coli
O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of
E. coli
O157:H7 by the addition of “nonfermentable” sugars and that this approach may be effective as a preharvest strategy for reducing carriage of
E. coli
O157:H7 in the rumen.
Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of
Proteus mirabilis
and
Escherichia coli
Authors: Hoischen Christian , Fritsche Christine , Gumpert Johannes , Westermann Martin , Gura Katleen , Fahnert Beatrix 
Published: February 2003

ABSTRACT

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of
Escherichia coli
and
Proteus mirabilis
. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from
E. coli
(lactose permease LacY, preprotein translocase SecY) and
P. mirabilis
(curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 μg ml
−1
, with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.
Osmoregulation and Fungicide Resistance: the
Neurospora crassa os-2
Gene Encodes a
HOG1
Mitogen-Activated Protein Kinase Homologue
Authors: Zhang Yan , Lamm Randy , Pillonel Christian , Lam Stephen , Xu Jin-Rong 
Published: February 2003

ABSTRACT

Neurospora crassa
osmosensitive (
os
) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that
os-2
and
os
-
5
mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the
os-2
gene by using sib selection.
os-2
encodes a putative mitogen-activated protein (MAP) kinase homologous to
HOG1
and can complement the osmosensitive phenotype of a
Saccharomyces cerevisiae hog1
mutant. We sequenced three
os-2
alleles and found that all of them were null with either frameshift or nonsense point mutations. An
os-2
gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely,
os-2
mutants transformed with the wild-type
os-2
gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in
os-2
mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of
N. crassa
is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive
os-2
MAP kinase pathway.
Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis
Authors: Bühler Bruno , Witholt Bernard , Hauer Bernhard , Schmid Andreas 
Published: February 2003

ABSTRACT

Xylene monooxygenase of
Pseudomonas putida
mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant
Escherichia coli
expressing the monooxygenase genes
xylM
and
xylA
catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using
E. coli
expressing
xylMA
, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of
E. coli
was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.
Sources and Species of
Cryptosporidium
Oocysts in the Wachusett Reservoir Watershed
Authors: Jellison Kristen L. , Hemond Harold F. , Schauer David B. 
Published: February 2003

ABSTRACT

Understanding the behavior of
Cryptosporidium
oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen
Cryptosporidium
parvum
from other
Cryptosporidium
species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.
l-Glucitol Catabolism inStenotrophomonas maltophiliaAc
Authors: Brechtel Elke , Huwig Alexander , Giffhorn Friedrich 
Published: February 2003

ABSTRACTThe carbohydrate catabolism of the bacteriumStenotrophomonas maltophiliaAc (previously namedPseudomonassp. strain Ac), which is known to convert the unnatural polyoll-glucitol tod-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channelsl-glucitol viad-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified.d-Sorbose was converted by C-3 epimerization tod-tagatose, which, in turn, was isomerized tod-galactose.d-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation ofd-galactose tod-galactonate, its dehydration to 2-keto-3-deoxy-d-galactonate, and its phosphorylation to 2-keto-3-deoxy-d-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate andd-glycerate 3-phosphate as the central metabolic intermediates.
Expression of ActA, Ami, InlB, and Listeriolysin O in
Listeria monocytogenes
of Human and Food Origin
Authors: Jacquet C. , Gouin E. , Jeannel D. , Cossart P. , Rocourt J. 
Published: February 2003

ABSTRACT

Expression of proteins involved in the adhesion of
Listeria monocytogenes
to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of
L. monocytogenes
strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90;
P
= 1 × 10
−4
). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89;
P
= 1 × 10
−2
) and central nervous system infections were associated with group ActA2 strains (OR = 3.04;
P
= 1 × 10
−3
) and group ActA3 strains (OR = 3.91;
P
= 1 × 10
−3
).
Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance
Authors: Di Cagno Raffaella , De Angelis Maria , Lavermicocca Paola , De Vincenzi Massimo , Giovannini Claudio , Faccia Michele , Gobbetti Marco 
Published: February 2003

ABSTRACT

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns,
Lactobacillus alimentarius
15M,
Lactobacillus brevis
14G,
Lactobacillus sanfranciscensis
7A, and
Lactobacillus hilgardii
51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by
L. alimentarius
15M,
L. brevis
14G, and
L. sanfranciscensis
7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of
L. alimentarius
15M and
L. brevis
14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.
Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from
Comamonas
sp. Strain JS765
Authors: Lessner Daniel J. , Johnson Glenn R. , Parales Rebecca E. , Spain Jim C. , Gibson David T. 
Published: February 2003

ABSTRACT

Comamonas
sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase
NBZ
, Ferredoxin
NBZ
, Oxygenase
NBZα
, and Oxygenase
NBZβ
, with the gene designations
nbzAa
,
nbzAb
,
nbzAc
, and
nbzAd
, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from
Pseudomonas
sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase
NBZ
revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.
Tetrachloroethene Dehalorespiration and Growth of
Desulfitobacterium frappieri
TCE1 in Strict Dependence on the Activity of
Desulfovibrio fructosivorans
Authors: Drzyzga Oliver , Gottschal Jan C. 
Published: February 2003

ABSTRACT

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium
Desulfovibrio fructosivorans
and the dehalorespiring
Desulfitobacterium frappieri
TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to
cis
-dichloroethene (
cis
-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to
cis
-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H
2
measurements of the gas phase and by the production of methane after the addition of a third organism,
Methanobacterium formicicum
.
Identification and Characterization of Pathogenic
Aeromonas veronii
Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh
Authors: Rahman Mokhlasur , Colque-Navarro Patricia , Kühn Inger , Huys Geert , Swings Jean , Möllby Roland 
Published: February 2003

ABSTRACT

Sparse information is available on the virulence factors of
Aeromonas
strains isolated from diseased fish, from the environment, and from humans. In the present study, 52
Aeromonas
isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be
Aeromonas veronii
biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi
Aeromonas
isolates. The 52
Aeromonas
isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of
A. veronii
biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.
Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea
Authors: Pernthaler Annelie , Preston Christina M. , Pernthaler Jakob , DeLong Edward F. , Amann Rudolf 
Published: February 2003

ABSTRACT

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (
Euryarchaeota
) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.
Characterization of the
norB
Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium
Synechocystis
sp. Strain PCC6803
Authors: Büsch Andrea , Friedrich Bärbel , Cramm Rainer 
Published: February 2003

ABSTRACT

A
norB
gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium
Synechocystis
sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier
Ralstonia
eutropha
. Heterologous complementation of a nitric oxide reductase-negative mutant of
R
.
eutropha
with
norB
from
Synechocystis
restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of
Synechocystis
wild-type cells. Virtually no nitric oxide reductase activity was present in a
norB
-negative mutant of
Synechocystis
. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for
Synechocystis
when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor
norB
expression in
Synechocystis
. Transcription of
norB
was not enhanced by the addition of the NO-generating agent sodium nitroprusside.