bluuebunny/crossref_metadata_2025 huggingface dataset:
Interplay between
Clostridium thermocellum
Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States
Authors: Vazana Yael , Moraïs Sarah , Barak Yoav , Lamed Raphael , Bayer Edward A.
Published: March 2010

The anaerobic, thermophilic cellulolytic bacterium
Clostridium thermocellum
is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from
C. thermocellum
. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and
vice versa
. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome.
C. thermocellum
is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.
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

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

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

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

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

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

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

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

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

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

Free 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

Members 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

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

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

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

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

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.
Contribution of Peroxisomes to Secondary Metabolism and Pathogenicity in the Fungal Plant Pathogen Alternaria alternata
Authors: Imazaki Ai , Tanaka Aiko , Harimoto Yoshiaki , Yamamoto Mikihiro , Akimitsu Kazuya , Park Pyoyun , Tsuge Takashi
Published: March 2010

The filamentous fungus
Alternaria alternata
includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes,
AKT1
,
AKT2
,
AKT3
, and
AKTR
, involved in AK toxin biosynthesis.
AKT1
,
AKT2
, and
AKT3
encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated Aa
PEX6
, which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made Aa
PEX6
disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAa
PEX6
mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAa
PEX6
mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAa
PEX6
mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in
A. alternata
. We also observed that mutation of Aa
PEX6
caused a marked reduction of conidiation.
Conserved Regulators of Mating Are Essential for Aspergillus fumigatus Cleistothecium Formation
Authors: Szewczyk Edyta , Krappmann Sven
Published: March 2010

Sexual reproduction of the human pathogen
Aspergillus fumigatus
(teleomorph:
Neosartorya fumigata
) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of
A. fumigatus
, and this evidence demonstrates the generality and reproducibility of this long-time-undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating-type idiomorphs
MAT1
-
1
and
MAT1
-
2
, as does expression of genes encoding factors presumably involved in this process. Moreover, analysis of an
A. fumigatus
mutant deleted for the
nsdD
gene suggests a role of this conserved regulator of cleistothecium development in hyphal fusion and hence heterokaryon formation.
Genome Sequence of
Pantoea ananatis
LMG20103, the Causative Agent of
Eucalyptus
Blight and Dieback
Authors: De Maayer Pieter , Chan Wai Yin , Venter Stephanus N. , Toth Ian K. , Birch Paul R. J. , Joubert Fourie , Coutinho Teresa A.
Published: March 2010

Pantoea ananatis
is a Gram-negative plant pathogen that causes disease on a broad range of host plants, including pineapple, maize, rice, onion, melons, and
Eucalyptus
, and has been implicated in several cases of human disease. Here, we report the genome sequence of
P. ananatis
LMG20103 isolated from diseased
Eucalyptus
in South Africa.
Genome Sequence of Naturally Competent
Aggregatibacter actinomycetemcomitans
Serotype a Strain D7S-1
Authors: Chen Casey , Kittichotirat Weerayuth , Chen Weizhen , Downey Jennifer S. , Si Yan , Bumgarner Roger
Published: March 2010

The major clonal lineages of the Gram-negative periodontal pathogen
Aggregatibacter actinomycetemcomitans
include serotype a, b, and c strains. Here, we report the draft genome sequence of a naturally competent serotype a strain, D7S-1, isolated from a patient with aggressive periodontitis.
Complete Genome Sequence of
Lactococcus lactis
subsp.
lactis
KF147, a Plant-Associated Lactic Acid Bacterium
Authors: Siezen Roland J. , Bayjanov Jumamurat , Renckens Bernadet , Wels Michiel , van Hijum Sacha A. F. T. , Molenaar Douwe , van Hylckama Vlieg Johan E. T.
Published: March 2010

Lactococcus lactis
is a lactic acid bacterium used in the production of many fermented dairy products. We report the complete genome sequence of
L. lactis
subsp.
lactis
KF147, a nondairy strain isolated from mung bean sprouts. The circular chromosome of 2,598,144 bp, the largest among the sequenced lactococcal strains, encodes many properties related to adaptation to the plant environment.
Identification ofChlamydia trachomatisOuter Membrane Complex Proteins by Differential Proteomics
Authors: Liu Xiaoyun , Afrane Mary , Clemmer David E. , Zhong Guangming , Nelson David E.
Published: March 2010

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses ofChlamydia trachomatisserovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.
The Sigma Factor AlgU Plays a Key Role in Formation of Robust Biofilms by NonmucoidPseudomonas aeruginosa
Authors: Bazire Alexis , Shioya Kouki , Soum-Soutéra Emmanuelle , Bouffartigues Emeline , Ryder Cynthia , Guentas-Dombrowsky Linda , Hémery Gaëlle , Linossier Isabelle , Chevalier Sylvie , Wozniak Daniel J. , Lesouhaitier Olivier , Dufour Alain
Published: March 2010

The extracytoplasmic function sigma factor AlgU ofPseudomonas aeruginosais responsible for alginate overproduction, leading to mucoidy and chronic infections of cystic fibrosis patients. We investigated here the role of AlgU in the formation of nonmucoid biofilms. ThealgUmutant ofP. aeruginosaPAO1 (PAOU) showed a dramatic impairment in biofilm formation under dynamic conditions. PAOU was defective both in cell attachment to glass and in development of robust, shear-resistant biofilms. This was explained by an impaired production of extracellular matrix, specifically of the exopolysaccharide Psl, as revealed by microscopy and enzyme-linked immunosorbent assay. Complementing thealgUmutation with a plasmid-bornealgUgene restored wild-type phenotypes. Compared with that in PAO1, expression of thepsloperon was reduced in the PAOU strain, and the biofilm formation ability of this strain was partially restored by inducing the transcription of thepsloperon. Furthermore, expression of the lectin-encodinglecAandlecBgenes was reduced in the PAOU strain. In agreement with the requirement of LecB for type IV pilus biogenesis, PAOU displayed impaired twitching motility. Collectively, these genetic downregulation events explain the biofilm formation defect of the PAOU mutant. Promoter mapping indicated that AlgU is probably not directly responsible for transcription of thepsloperon and thelecgenes, but AlgU is involved in the expression of theppyRgene, whose product was reported to positively controlpslexpression. Expressing theppyRgene in PAOU partially restored the formation of robust biofilms.
ON THE EFFICIENT COMPUTATION OF THE MINIMAL COVERABILITY SET OF PETRI NETS
Authors: GEERAERTS GILLES , RASKIN JEAN-FRANÇOIS , VAN BEGIN LAURENT
Published: March 2010

The minimal coverability set (MCS) of a Petri net is a finite representation of the downward-closure of its reachable markings. The minimal coverability set allows to decide several important problems like coverability, semi-liveness, place boundedness, etc. The classical algorithm to compute the MCS constructs the Karp&Miller (KM) tree [10]. Unfortunately the KM tree is often huge, even for small nets. An improvement of this KM algorithm is the Minimal Coverability Tree (MCT) algorithm [3], which has been introduced nearly 20 years ago, and implemented since then in several tools such as Pep [9]. Unfortunately, we show in this paper that the MCT is flawed: it might compute an under-approximation of the reachable markings. We propose a new solution for the efficient computation of the MCS of Petri nets. Our algorithm is based on new ideas, and the experimental results show that it behaves much better in practice than the KM algorithm.
LATTICED SIMULATION RELATIONS AND GAMES
Authors: KUPFERMAN ORNA , LUSTIG YOAD
Published: March 2010

Multi-valued Kripke structures are Kripke structures in which the atomic propositions and the transitions are not Boolean and can take values from some set. In particular, latticed Kripke structures, in which the elements in the set are partially ordered, are useful in abstraction, query checking, and reasoning about multiple view-points. The challenges that formal methods involve in the Boolean setting are carried over, and in fact increase, in the presence of multi-valued systems and logics. We lift to the latticed setting two basic notions that have been proven useful in the Boolean setting. We first define latticed simulation between latticed Kripke structures. The relation maps two structures M1 and M2 to a lattice element that essentially denotes the truth value of the statement "every behavior of M1 is also a behavior of M2". We show that latticed-simulation is logically characterized by the universal fragment of latticed µ-calculus, and can be calculated in polynomial time. We then proceed to defining latticed two-player games. Such games are played along graphs in which each transition have a value in the lattice. The value of the game essentially denotes the truth value of the statement "the ∨-player can force the game to computations that satisfy the winning condition". An earlier definition of such games involved a zig-zagged traversal of paths generated during the game. Our definition involves a forward traversal of the paths, and it leads to better understanding of multi-valued games. In particular, we prove a min-max property for such games, and relate latticed simulation with latticed games.
EFFICIENT, ALMOST EXACT SIMULATION OF THE HESTON STOCHASTIC VOLATILITY MODEL
Authors: VAN HAASTRECHT ALEXANDER , PELSSER ANTOON
Published: March 2010

We deal with discretization schemes for the simulation of the Heston stochastic volatility model. These simulation methods yield a popular and flexible pricing alternative for pricing and managing a book of exotic derivatives which cannot be valued using closed-form expressions. For the Heston dynamics an exact simulation method was developed by Broadie and Kaya (2006), however we argue why its practical use is limited. Instead we focus on efficient approximations of the exact scheme, aimed to resolve the disadvantages of this method; one of the main bottlenecks in the exact scheme is the simulation of the Non-central Chi-squared distributed variance process, for which we suggest an efficient caching technique. At first sight the creation of a cache containing the inverses of this distribution might seem straightforward, however as the parameter space of the inverse Non-central Chi-squared distribution is three-dimensional, the design of such a direct cache is rather complicated, as pointed out by Broadie and Andersen. Nonetheless, for the case of the Heston model we are able to tackle this dimensionality problem and show that the three-dimensional inverse of the non-central chi-squared distribution can effectively be reduced to a one dimensional cache. The performed analysis hence leads to the development of three new efficient simulation methods (the NCI, NCI-QE and BK-DI scheme). Finally, we conclude with a comprehensive numerical study of these new schemes and the exact scheme of Broadie and Kaya, the almost exact scheme of Smith, the Kahl-Jäckel scheme, the FT scheme of Lord et al. and the QE-M scheme of Andersen. From these results, we find that the QE-M scheme is the most efficient, followed closely by the NCI-M, NCI-QE-M and BK-DI-M schemes, whilst we observe that all other considered schemes perform a factor 6 to 70 times less efficient than the latter four methods.
PRICING CMS SPREAD OPTIONS IN A LIBOR MARKET MODEL
Authors: BELOMESTNY DENIS , KOLODKO ANASTASIA , SCHOENMAKERS JOHN
Published: March 2010

We present two approximation methods for the pricing of CMS spread options in Libor market models. Both approaches are based on approximating the underlying swap rates with lognormal processes under suitable measures. The first method is derived straightforwardly from the Libor market model. The second one uses a convexity adjustment technique under a linear swap model assumption. A numerical study demonstrates that both methods provide satisfactory approximations of spread option prices and can be used for calibration of a Libor market model to the CMS spread option market.
IMPLICATION OF THE KELLY CRITERION FOR MULTI-DIMENSIONAL PROCESSES
Authors: LV YINGDONG , MEISTER BERNHARD K.
Published: March 2010

In this paper, we study the Kelly criterion in the continuous time framework building on the work of E.O. Thorp and others. The existence of an optimal strategy is proven in a general setting and the corresponding optimal wealth process is found. A simple formula is provided for calculating the optimal portfolio in terms of drift, short term risk-free rate and correlations for a set of generic multi-dimensional diffusion processes satisfying some simple conditions. Properties of the optimal investment strategy are studied. The paper ends with a short discussion of the implications of these ideas for financial markets.
THE IMPORTANCE OF INHIBITORS FOR THE SIMULATION OF METABOLIC PROCESSES: IN SILICOZn2+ INHIBITION OF m-ACONITASE FROM ANALYSIS OF GLYCOLYSIS AND KREBS CYCLE KINETIC MODELS
Authors: ČUPERLOVIĆ-CULF MIROSLAVA
Published: March 2010

Metal ions have a major effect on the metabolic processes in cells either as inhibitors or as integral components of enzymes. The inhibition of enzymes can take place either through the inhibition of gene expression or through inhibition of protein function. A particularly interesting example of the effect of a metal ion on metabolism is the observed inhibition of Krebs cycle and alteration of energy metabolism by zinc (II) cations. In this particular case metal ion inhibition of enzyme is linked to one of the major functions of prostate cells of accumulation and excretion of citrate. Experimental results have shown that increase in concentration of zinc (II) in prostate cells effectively blocks progression of a part of the Krebs cycle leading to change in the concentration of several metabolites with largest effect in the accumulation of citrate. Based on previously reported experimental results, several distinct mechanisms for zinc (II) inhibition of Krebs cycle were proposed. In order to determine the precise mechanism of inhibition in this system, a mathematical model of glycolysis and Krebs cycle was constructed. Three different types of inhibition were analyzed, including competitive and uncompetitive inhibition as well as inhibition through the alteration of the expression level of m-aconitase. The effects of different inhibition models on metabolite concentrations were investigated as a time course simulation of the system of reactions. Several kinetic parameters in the model were optimized in order to best resemble experimental measurements. The simulation shows that only competitive inhibition leads to an agreement with experimental data.
Tuned Range-Separated Hybrids in Density Functional Theory
Authors: Baer Roi , Livshits Ester , Salzner Ulrike
Published: March 2010

We review density functional theory (DFT) within the Kohn-Sham (KS) and the generalized KS (GKS) frameworks from a theoretical perspective for both time-independent and time-dependent problems. We focus on the use of range-separated hybrids within a GKS approach as a practical remedy for dealing with the deleterious long-range self-repulsion plaguing many approximate implementations of DFT. This technique enables DFT to be widely relevant in new realms such as charge transfer, radical cation dimers, and Rydberg excitations. Emphasis is put on a new concept of system-specific range-parameter tuning, which introduces predictive power in applications considered until recently too difficult for DFT.
Hyper-Raman Scattering by Molecular Vibrations
Authors: Kelley Anne Myers
Published: March 2010

This article reviews the experimental and theoretical aspects of vibrational hyper-Raman scattering from molecules. Particular emphasis is placed on hyper-Raman scattering enhanced by nanostructured metal surfaces and by two-photon electronic resonance.
Molecular Structural Dynamics Probed by Ultrafast X-Ray Absorption Spectroscopy
Authors: Bressler Christian , Chergui Majed
Published: March 2010

The ability to visualize molecular structure in the course of a chemical reaction or a biological function has been a dream of scientists for decades. X-ray absorption spectroscopy (XAS) is ideal in this respect because it is chemically selective and can be implemented in any type of medium. Furthermore, using X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) in laser pump/X-ray probe experiments allows the retrieval of not only the local geometric structure of the system under study, but also the underlying electronic structure changes that drive the structural dynamics. We review recent developments in picosecond and femtosecond XAS applied to molecular systems in solution. Examples on ultrafast photoinduced processes such as intramolecular electron transfer, low-to-high spin change, and bond formation are presented.
Chemistry of Hofmeister Anions and Osmolytes
Authors: Zhang Yanjie , Cremer Paul S.
Published: March 2010

The study of the interactions of salts and osmolytes with macromolecules in aqueous solution originated with experiments concerning protein precipitation more than 100 years ago. Today, these solutes are known to display recurring behavior for myriad biological and chemical processes. Such behavior depends both on the nature and concentration of the species in solution. Despite the generality of these effects, our understanding of the molecular-level details of ion and osmolyte specificity is still quite limited. Here, we review recent studies of the interactions between anions and urea with model macromolecular systems. A mechanism for specific ion effects is elucidated for aqueous systems containing charged and uncharged polymers, polypeptides, and proteins. The results clearly show that the effects of the anions are local and involve direct interactions with macromolecules and their first hydration shell. Also, a hydrogen-bonding mechanism is tested for the urea denaturation of proteins with some of these same systems. In that case, direct hydrogen bonding can be largely discounted as the key mechanism for urea stabilization of uncollapsed and/or unfolded structures.
Intermittent pneumatic leg compressions acutely upregulate VEGF and MCP-1 expression in skeletal muscle
Authors: Roseguini Bruno T. , Mehmet Soylu S. , Whyte Jeffrey J. , Yang H. T. , Newcomer Sean , Laughlin M. Harold
Published: March 2010

Application of intermittent pneumatic compressions (IPC) is an extensively used therapeutic strategy in vascular medicine, but the mechanisms by which this method works are unclear. We tested the hypothesis that acute application (150 min) of cyclic leg compressions in a rat model signals upregulation of angiogenic factors in skeletal muscle. To explore the impact of different pressures and frequency of compressions, we divided rats into four groups as follows: 120 mmHg (2 s inflation/2 s deflation), 200 mmHg (2 s/2 s), 120 mmHg (4 s/16 s), and control (no intervention). Blood flow and leg oxygenation ( study 1) and the mRNA expression of angiogenic mediators in the rat tibialis anterior muscle ( study 2) were assessed after a single session of IPC. In all three groups exposed to the intervention, a modest hyperemia (∼37% above baseline) between compressions and a slight, nonsignificant increase in leg oxygen consumption (∼30%) were observed during IPC. Compared with values in the control group, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) mRNA increased significantly ( P < 0.05) only in rats exposed to the higher frequency of compressions (2 s on/2 s off). Endothelial nitric oxide synthase, matrix metalloproteinase-2, and hypoxia-inducible factor-1α mRNA did not change significantly following the intervention. These findings show that IPC application augments the mRNA content of key angiogenic factors in skeletal muscle. Importantly, the magnitude of changes in mRNA expression appeared to be modulated by the frequency of compressions such that a higher frequency (15 cycles/min) evoked more robust changes in VEGF and MCP-1 compared with a lower frequency (3 cycles/min).
α1-Adrenergic receptor subtype function in fetal and adult cerebral arteries
Authors: Goyal Ravi , Mittal Ashwani , Chu Nina , Zhang Lubo , Longo Lawrence D.
Published: March 2010

In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of α1-adrenergic receptor (α1-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of α1-AR subtypes. In CA from fetal (∼140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular Ca2+concentration ([Ca2+]i), radioimmunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. The α1A-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [Ca2+]iincrease was reduced significantly in both age groups. The α1D-AR antagonist (BMY-7378) blocked both Phe-induced contractions and Ca2+responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of α1A-AR and α1B-AR, but not α1D-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the α1-AR subtype expression was only ∼20% in fetal CA compared with the adult. Moreover, in fetal CA, the α1D-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by α1B-AR (CEC) and α1D-AR (BMY-7378) inhibitors, but not by α1A-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, α1B-AR and α1D-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA α1B-AR and α1D-AR subtypes may be a critical factor associated with cerebrovascular growth and function.