bluuebunny/crossref_metadata_2025_split huggingface dataset:
PCR-Denaturing Gradient Gel Electrophoresis Analysis To Assess the Effects of a Genetically Modified Cucumber Mosaic Virus-Resistant Tomato Plant on Soil Microbial Communities
Authors: Lin Chih-Hui , Pan Tzu-Ming 
Published: March 2010

ABSTRACT
The effects of a genetically modified cucumber mosaic virus (CMV)-resistant tomato on soil microbial communities were evaluated in this study. Soil position and environmental factors played more dominant roles than the tomato genotype in the variation of soil microbial communities.
In Silico
Identification of Gene Amplification Targets for Improvement of Lycopene Production
Authors: Choi Hyung Seok , Lee Sang Yup , Kim Tae Yong , Woo Han Min 
Published: March 2010

ABSTRACT

The identification of genes to be deleted or amplified is an essential step in metabolic engineering for strain improvement toward the enhanced production of desired bioproducts. In the past, several methods based on flux analysis of genome-scale metabolic models have been developed for identifying gene targets for deletion. Genome-wide identification of gene targets for amplification, on the other hand, has been rather difficult. Here, we report a strategy called
f
lux
s
canning based on
e
nforced
o
bjective
f
lux (FSEOF) to identify gene amplification targets. FSEOF scans all the metabolic fluxes in the metabolic model and selects fluxes that increase when the flux toward product formation is enforced as an additional constraint during flux analysis. This strategy was successfully employed for the identification of gene amplification targets for the enhanced production of the red-colored antioxidant lycopene. Additional metabolic engineering based on gene knockout simulation resulted in further synergistic enhancement of lycopene production. Thus, FSEOF can be used as a general strategy for selecting genome-wide gene amplification targets
in silico
.
Subsurface Cycling of Nitrogen and Anaerobic Aromatic Hydrocarbon Biodegradation Revealed by Nucleic Acid and Metabolic Biomarkers
Authors: Yagi Jane M. , Suflita Joseph M. , Gieg Lisa M. , DeRito Christopher M. , Jeon Che-Ok , Madsen Eugene L. 
Published: March 2010

ABSTRACT

Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore
in situ
field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome
c
nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred
in situ
. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to
in situ
cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).
ArcS, the Cognate Sensor Kinase in an Atypical Arc System of
Shewanella oneidensis
MR-1
Authors: Lassak Jürgen , Henche Anna-Lena , Binnenkade Lucas , Thormann Kai M. 
Published: March 2010

ABSTRACT

The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as
Shewanella
species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of
Escherichia coli
, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in
Shewanella
, and as in
Escherichia coli
,
Shewanella
ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in
Shewanella oneidensis
MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the
Shewanella
Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.
Risk of
Listeria monocytogenes
Contamination of Raw Ready-To-Eat Seafood Products Available at Retail Outlets in Japan
Authors: Miya Satoko , Takahashi Hajime , Ishikawa Tatsuya , Fujii Tateo , Kimura Bon 
Published: March 2010

ABSTRACT

Examination of
Listeria monocytogenes
prevalence among ready-to-eat foods in Japan revealed frequent (5.7 to 12.1%) contamination of minced tuna and fish roe products, and the isolates had the same virulence levels as clinical isolates in terms of invasion efficiency and infectivity in cell cultures and a murine infection model, respectively. Premature stop codons in
inlA
were infrequent (1 out of 39 isolates). Cell numbers of
L. monocytogenes
in minced tuna and salmon roe increased rapidly under inappropriate storage temperatures (from a most probable number [MPN] of 10
0
to 10
1
/g to an MPN of 10
3
to 10
4
/g over the course of 2 days at 10°C). Thus, regulatory guidelines are needed for acceptable levels of
L. monocytogenes
in these foods.
Efficient Electrotransformation of
Bacteroides fragilis
Authors: Ichimura Minoru , Nakayama-Imaohji Haruyuki , Wakimoto Shin , Morita Hidetoshi , Hayashi Tetsuya , Kuwahara Tomomi 
Published: March 2010

ABSTRACT

This study describes refined electroporation parameters for efficient transformation of
Bacteroides fragilis
by plasmids prepared from laboratory strains of
Escherichia coli
. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four
E. coli
-
Bacteroides
shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for
B. fragilis
transformation, and it generated 2- to 900-fold more transformants (about 10
4
transformants per μg pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested,
B. fragilis
cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of
B. fragilis
genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in
B. fragilis
strain NCTC9343 only when a homologously prepared (i.e.,
in vivo
methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a
B. fragilis
gene using
in vivo
-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of
Bacteroides
species.
Relative Decay of
Bacteroidales
Microbial Source Tracking Markers and Cultivated
Escherichia coli
in Freshwater Microcosms
Authors: Dick Linda K. , Stelzer Erin A. , Bertke Erin E. , Fong Denise L. , Stoeckel Donald M. 
Published: March 2010

ABSTRACT

Fecal indicator bacteria (FIB), commonly used to regulate sanitary water quality, cannot discriminate among sources of contamination. The use of alternative quantitative PCR (qPCR) methods for monitoring fecal contamination or microbial source tracking requires an understanding of relationships with cultivated FIB, as contamination ages under various conditions in the environment. In this study, the decay rates of three
Bacteroidales
16S rRNA gene markers (AllBac for general contamination and qHF183 and BacHum for human-associated contamination) were compared with the decay rate of cultivated
Escherichia coli
in river water microcosms spiked with human wastewater. The following five sets of microcosms were monitored over 11 days: control, artificial sunlight, sediment exposure, reduced temperature, and no autochthonous predation. Decay was characterized by estimation of the time needed to produce a 2-log reduction (
t
99
). No treatment-associated differences in the decay of the 4 targets were evident except with reduced predation, where
E. coli
, qHF183, and BacHum markers had lower levels of decay by day 3. However, there were substantial target-associated differences. Decay curves for the AllBac marker indicated a larger persistent population than those of the other targets. Exposure to sunlight, sediment, and reduced predation resulted in more rapid decay of the human-associated markers relative to cultivable
E. coli
, but there were no differences in
t
99
values among the 4 targets under control conditions or at reduced temperatures. Further evaluation of epidemiological relationships will be needed in order to relate the markers directly to health risk. These findings suggest that the tested human-associated markers can complement
E. coli
as indicators of the human impact on sanitary water quality under the constrained conditions described in this paper.
Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil
Authors: İnceoǧlu Özgül , Hoogwout Eelco F. , Hill Patrick , van Elsas Jan Dirk 
Published: March 2010

ABSTRACT
Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods. Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.
Considerable Increase in Resveratrol Production by Recombinant Industrial Yeast Strains with Use of Rich Medium
Authors: Sydor Tobias , Schaffer Steffen , Boles Eckhard 
Published: March 2010

ABSTRACT

Resveratrol synthesis from
p
-coumarate was analyzed in different
Saccharomyces cerevisiae
strains expressing the 4-coumaroyl-coenzyme A ligase (4CL1) from
Arabidopsis thaliana
and the stilbene synthase (STS) from
Vitis vinifera
and compared between yeast cultures growing in rich or synthetic medium. The use of rich medium considerably improved resveratrol production, and resveratrol yields of up to 391 mg/liter could be achieved with an industrial Brazilian sugar cane-fermenting yeast.
The Transcriptional Repressor FarR Is Not Involved in Meningococcal Fatty Acid Resistance Mediated by the FarAB Efflux Pump and Dependent on Lipopolysaccharide Structure
Authors: Schielke Stephanie , Schmitt Corinna , Spatz Carolin , Frosch Matthias , Schubert-Unkmeir Alexandra , Kurzai Oliver 
Published: March 2010

ABSTRACTFree fatty acids are important antimicrobial substances regulating the homeostasis of colonizing bacteria on epithelial surfaces. Here, we show that meningococci express a functionalfarABefflux pump, which is indispensable for fatty acid resistance. However, other than inNeisseria gonorrhoeae, the transcriptional regulator FarR is not involved in regulation of this operon inNeisseria meningitidis. We tested the susceptibility of 23 meningococcal isolates against saturated and unsaturated long-chain fatty acids, proving that meningococci are generally highly resistant, with the exception of serogroup Y strains belonging to sequence type 23. Using genetically determined lipopolysaccharide (LPS)-truncated mutant strains, we show that addition of the LPS core oligosaccharide and hexa-acylation of its membrane anchor lipid A are imperative for fatty acid resistance of meningococci. The sensitivity of the serogroup Y strains is due to naturally occurring mutations within thelpxL1gene, which is responsible for addition of the sixth acyl chain on the LPS membrane anchor lipid A. Therefore, fatty acid resistance in meningococci is provided by both the active efflux pump FarAB and by the natural permeability barrier of the Gram-negative outer membrane. The transcriptional regulator FarR is not implicated in fatty acid resistance in meningococci, possibly giving rise to a constitutively active FarAB efflux pump system and thus revealing diverse mechanisms of niche adaptation in the two closely relatedNeisseriaspecies.
Characterization of the Serpin-Encoding Gene ofBifidobacterium breve210B
Authors: Turroni Francesca , Foroni Elena , O'Connell Motherway Mary , Bottacini Francesca , Giubellini Vanessa , Zomer Aldert , Ferrarini Alberto , Delledonne Massimo , Zhang Ziding , van Sinderen Douwe , Ventura Marco 
Published: March 2010

ABSTRACTMembers of the serpin (serineproteaseinhibitor) superfamily have been identified in higher multicellular eukaryotes, as well as in bacteria, although examination of available genome sequences has indicated that homologs of the bacterial serpin-encoding gene (ser) are not widely distributed. In members of the genusBifidobacteriumthis gene appears to be present in at least 5, and perhaps up to 9, of the 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria produce serpins that form a separate clade. We characterized theser210Blocus ofBifidobacterium breve210B, which encompasses a number of genes whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, microarray, reverse transcription-PCR (RT-PCR), and quantitative real-time PCR (qRT-PCR) analyses revealed that a 3.5-kb polycistronic mRNA encompassing theser210Boperon with a single transcriptional start site is strongly induced following treatment ofB. breve210B cultures with some proteases. Interestingly, transcription of other bifidobacterialserhomologs appears to be triggered by different proteases.
Proteomic Analyses To Reveal the Protective Role of Glutathione in Resistance of
Lactococcus lactis
to Osmotic Stress
Authors: Zhang Yanhe , Zhang Yanping , Zhu Yan , Mao Shaoming , Li Yin 
Published: March 2010

ABSTRACT

Previously, we have shown that glutathione can protect
Lactococcus lactis
against oxidative stress and acid stress. In this study, we show that glutathione taken up by
L. lactis
SK11 can protect this organism against osmotic stress. When exposed to 5 M NaCl,
L. lactis
SK11 cells containing glutathione exhibited significantly improved survival compared to the control cells. Transmission electron microscopy showed that the integrity of
L. lactis
SK11 cells containing glutathione was maintained for at least 24 h, whereas autolysis of the control cells occurred within 2 h after exposure to this osmotic stress. Comparative proteomic analyses using SK11 cells containing or not containing glutathione that were exposed or not exposed to osmotic stress were performed. The results revealed that 21 of 29 differentially expressed proteins are involved in metabolic pathways, mainly sugar metabolism. Several glycolytic enzymes of
L. lactis
were significantly upregulated in the presence of glutathione, which might be the key for improving the general stress resistance of a strain. Together with the results of previous studies, the results of this study demonstrated that glutathione plays important roles in protecting
L. lactis
against multiple environmental stresses; thus, glutathione can be considered a general protectant for improving the robustness and stability of dairy starter cultures.
Construction of Aminoglycoside-Sensitive
Burkholderia cenocepacia
Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay
Authors: Hamad Mohamad A. , Skeldon Alexander M. , Valvano Miguel A. 
Published: March 2010

ABSTRACT

Burkholderia cenocepacia
is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by
B. cenocepacia
. Here, we report the construction of gentamicin-sensitive strains of
B. cenocepacia
carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by
B. cenocepacia
, which can replicate in RAW 264.7 macrophages.
Expression in
Escherichia coli
of the Native
cyt1Aa
from
Bacillus thuringiensis
subsp.
israelensis
Authors: Sazhenskiy Vladislav , Zaritsky Arieh , Itsko Mark 
Published: March 2010

ABSTRACT

The gene
cyt1Aa
is one of the genes in the complex determining the mosquito larvicidity of
Bacillus thuringiensis
subsp.
israelensis
. Previous cloning in
Escherichia coli
resulted in a 48-bp addition upstream, encoding a chimera. Here,
cyt1Aa
was recloned without the artifact, and its toxicity against
Aedes aegypti
larvae and host
E. coli
cells was retested.
Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p
Authors: Basso Luiz R. , Gast Charles E. , Mao Yuxin , Wong Brian 
Published: March 2010

ABSTRACT

A major cause of azole resistance in
Candida albicans
is overexpression of
CDR1
,
CDR2
, and/or
MDR1
, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used
ACT1
- and
GAL1
-regulated expression plasmids to overexpress
CDR1
,
CDR2
, or
MDR1
in a
C. albicans
cdr1 cdr2 mdr1
-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [
3
H]fluconazole intracellularly, than empty-vector controls. Next, we used a
GAL1
-regulated dominant negative
sec4
allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from
CDR1-
,
CDR2
-, or
MDR1-
overexpressing cells accumulated much more [
3
H]fluconazole than did PGVs from empty-vector controls. The

K
m

s (expressed in micromolar concentrations) and
V
max
s (expressed in picomoles per milligram of protein per minute), respectively, for [
3
H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [
3
H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [
3
H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [
3
H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC
50
s; expressed as proportions of the [
3
H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in
C. albicans
and other medically important fungi.
Characterization of the Roco Protein Family in Dictyostelium discoideum
Authors: van Egmond Wouter N. , van Haastert Peter J. M. 
Published: March 2010

ABSTRACT

The Roco family consists of multidomain Ras-GTPases that include LRRK2, a protein mutated in familial Parkinson's disease. The genome of the cellular slime mold
Dictyostelium discoideum
encodes 11 Roco proteins. To study the functions of these proteins, we systematically knocked out the
roco
genes. Previously described functions for GbpC, Pats1, and QkgA (Roco1 to Roco3) were confirmed, while novel developmental defects were identified in
roco4
- and
roco11
-null cells. Cells lacking Roco11 form larger fruiting bodies than wild-type cells, while
roco4
-null cells show strong developmental defects during the transition from mound to fruiting body; prestalk cells produce reduced levels of cellulose, leading to unstable stalks that are unable to properly lift the spore head. Detailed phylogenetic analysis of four slime mold species reveals that QkgA and Roco11 evolved relatively late by duplication of an ancestor
roco4
gene (later than ∼300 million years ago), contrary to the situation with other
roco
genes, which were already present before the split of the common ancestor of
D. discoideum
and
Polysphondylium pallidum
(before ∼600 million years ago). Together, our data show that the
Dictyostelium
Roco proteins serve a surprisingly diverse set of functions and highlight Roco4 as a key protein for proper stalk cell formation.