bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence, Source, and Human Infection Potential of Cryptosporidium and Giardia spp. in Source and Tap Water in Shanghai, China
Authors: Feng Yaoyu , Zhao Xukun , Chen Jiaxu , Jin Wei , Zhou Xiaonong , Li Na , Wang Lin , Xiao Lihua
Published: April 2011

Genotyping studies on the source and human infection potential of
Cryptosporidium
oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for
Cryptosporidium
and 18% for
Giardia
by Method 1623, whereas 22% were positive for
Cryptosporidium
and 10% for
Giardia
by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of
Cryptosporidium
(28%) was similar to that obtained by Method 1623. Genotyping of
Cryptosporidium
in 17 water samples identified the presence of
C. andersoni
in 14 water samples,
C. suis
in 7 water samples,
C. baileyi
in 2 water samples,
C. meleagridis
in 1 water sample, and
C. hominis
in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans.
Cryptosporidium
oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of
Cryptosporidium
contamination in water, although the results from this method are semiquantitative.
Identification of Nitrogen-Incorporating Bacteria in Petroleum-Contaminated Arctic Soils by Using [
15
N]DNA-Based Stable Isotope Probing and Pyrosequencing
Authors: Bell Terrence H. , Yergeau Etienne , Martineau Christine , Juck David , Whyte Lyle G. , Greer Charles W.
Published: April 2011

Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil
in situ
to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [
15
N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (
alkB
) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and
alkB
gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of
Proteobacteria
and
Actinobacteria
that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of
Sphingomonadaceae
that was most enriched in
15
N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.
Grape Seed Extract for Control of Human Enteric Viruses
Authors: Su Xiaowei , D'Souza Doris H.
Published: April 2011

Grape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ∼7 log
10
PFU/ml or ∼5 log
10
PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (∼7 log
10
PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log
10
PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log
10
PFU/ml; MS2 by 1.13, 1.43, and 1.60 log
10
PFU/ml; and HAV by 1.81, 2.66, and 3.20 log
10
PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (
P
< 0.05) in a dose-dependent manner. GSE treatment of low titers (∼5 log
10
PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.
Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module
Authors: Sakka Makiko , Higashi Yurika , Kimura Tetsuya , Ratanakhanokchai Khanok , Sakka Kazuo
Published: April 2011

Paenibacillus curdlanolyticus
B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.
Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces
Authors: Peterson Robyn , Grinyer Jasmine , Nevalainen Helena
Published: April 2011

Coprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization–time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography–tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungus
Doratomyces stemonitis
C8 (EU551185) isolated from koala feces. As the genome of
D. stemonitis
has not been sequenced, cross-species identification,
de novo
sequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in the
D. stemonitis
C8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploited
T. reesei
. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.
Comparison of the Specificities and Efficacies of Primers for Aromatic Dioxygenase Gene Analysis of Environmental Samples
Authors: Iwai Shoko , Johnson Timothy A. , Chai Benli , Hashsham Syed A. , Tiedje James M.
Published: April 2011

Aromatic dioxygenase genes have long been of interest for bioremediation and aromatic carbon cycling studies. To date, 115 primers and probes have been designed and used to analyze dioxygenase gene diversities in environmental samples. Here we analyze those primers' specificities, coverages, and PCR product lengths compared to known aromatic dioxygenase genes based on
in silico
analysis as well as summarize their differing advantages or effectiveness from over 50 reported experimental studies. We also provide some guidance for primer use in future studies.
Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification
Authors: Chen Siyi , Wang Fei , Beaulieu John C. , Stein Rebecca E. , Ge Beilei
Published: April 2011

Recent outbreaks linked to
Salmonella
-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting
Salmonella
. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed
Salmonella
cells with concentrations up to 10
8
CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable
Salmonella
cells in pure culture and 6.1 × 10
3
to 6.1 × 10
4
CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (

T
T

) values and viable
Salmonella
cell numbers was high (
R
2
= 0.949 to 0.993), with a quantification range (10
2
to 10
5
CFU/reaction in pure culture and 10
4
to 10
7
CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable
Salmonella
cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.
Diversity of Culturable Psychrophilic and Psychrotrophic Anaerobic Bacteria Isolated from Beef Abattoirs and Their Environments
Authors: Moschonas G. , Bolton D. J. , McDowell D. A. , Sheridan J. J.
Published: April 2011

This study identified 431 psychrophilic or psychrotrophic isolates from commercial Irish beef abattoir environments and “blown packs” of vacuum-packed beef, using PCR and 16S rRNA sequencing, and estimated their intraspecies genetic diversity using restriction fragment length polymorphism (RFLP) analysis and spacer region PCR (SR-PCR). Twenty-five species were identified in the 431 isolates, with the most frequently recovered species being
Clostridium gasigenes
(
n
= 315),
Clostridium estertheticum
(
n
= 17), and a potentially novel species designated strain TC1 (
n
= 52). These species were previously found to be associated with a particular type of spoilage known as blown-pack spoilage (BPS), which occurs in chilled-stored (i.e., −1.5°C to 4°C) vacuum-packaged meat within 2 to 4 weeks and involves the production of large volumes of gas. Overall, the study demonstrates the considerable and not previously reported diversity of the anaerobic microflora in abattoirs and the presence of a wide range of organisms capable of causing BPS at chilled temperatures.
Microarray-Based Evaluation of Whole-Community Genome DNA Amplification Methods
Authors: Wang Jian , Van Nostrand Joy D. , Wu Liyou , He Zhili , Li Guanghe , Zhou Jizhong
Published: April 2011

Three whole-community genome amplification methods,
Bst
, REPLI-g, and Templiphi, were evaluated using a microarray-based approach. The amplification biases of all methods were <3-fold. For pure-culture DNA, REPLI-g and Templiphi showed less bias than
Bst
. For community DNA, REPLI-g showed the least bias and highest number of genes, while
Bst
had the highest success rate and was suitable for low-quality DNA.
Dynamics of Microbial Community Composition and Function during In Situ Bioremediation of a Uranium-Contaminated Aquifer
Authors: Van Nostrand Joy D. , Wu Liyou , Wu Wei-Min , Huang Zhijian , Gentry Terry J. , Deng Ye , Carley Jack , Carroll Sue , He Zhili , Gu Baohua , Luo Jian , Criddle Craig S. , Watson David B. , Jardine Philip M. , Marsh Terence L. , Tiedje James M. , Hazen Terry C. , Zhou Jizhong
Published: April 2011

A pilot-scale system was established to examine the feasibility of
in situ
U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter
−1
). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.
Functional Analysis of
ycfR
and
ycfQ
in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce
Authors: Deng Kaiping , Wang Siyun , Rui Xiaoqian , Zhang Wei , Tortorello Mary Lou
Published: April 2011

Fresh produce has been associated with multiple outbreaks of illness caused by
Escherichia coli
O157:H7. The mechanism of

E. coli

O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that
ycfR
was the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator in

E. coli

K-12 strains; however, its role in the pathogenic

E. coli

O157:H7 has not been clearly defined. In this study,
ycfR
was replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolar
ycfR
::
cat
mutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and the
ycfR
mutants. Both polar and nonpolar
ycfR
mutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion of
ycfR
in TW14359 did not change chlorine resistance, indicating that
ycfR
in these two outbreak-related

E. coli

O157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolar
ycfR
mutant exhibited lower survival compared to the wild type. The results suggest a role for
ycfR
in survival of Sakai during chlorine exposure. We also found that the upstream
ycfQ
, which is annotated as a DNA-binding regulator, acted as a repressor of
ycfR
. These findings suggest that gene regulation may be a mechanism by which

E. coli

O157:H7 strain Sakai could survive in the postharvest processing environment.
Facilitation of Bacterial Adaptation to Chlorothalonil-Contaminated Sites by Horizontal Transfer of the Chlorothalonil Hydrolytic Dehalogenase Gene
Authors: Liang Bin , Wang Guangli , Zhao Yanfu , Chen Kai , Li Shunpeng , Jiang Jiandong
Published: April 2011

Horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene (
chd
) is proposed based on the high conservation of the
chd
gene and its close association with a novel insertion sequence, IS
Ocsp1
, in 16 isolated chlorothalonil-dechlorinating strains belonging to eight different genera. The ecological role of horizontal gene transfer is assumed to facilitate bacterial adaptation to chlorothalonil-contaminated sites, through detoxification of chlorothalonil to less toxic 2,4,5-trichloro-6-hydroxybenzene-1,3-dicarbonitrile.
Development and Validation of a Concentration Method for the Detection of Influenza A Viruses from Large Volumes of Surface Water
Authors: Deboosere Nathalie , Horm Srey Viseth , Pinon Anthony , Gachet Jessica , Coldefy Chloé , Buchy Philippe , Vialette Michèle
Published: April 2011

Contamination of lakes and ponds plays an essential role as a reservoir of avian influenza A virus (AIV) in the environment. A method to concentrate waterborne AIV is a prerequisite for the detection of virus present at low levels in water. The aim of this study was to develop and validate a method for the concentration and detection of infectious AIV from large volumes of surface water samples. Two filtration systems, glass wool and electropositive NanoCeram filter, were studied. The individual effects of filtration-elution and polyethylene glycol (PEG) concentration parameters on the recovery efficiency of the H1N1 strain from 10-liter surface water samples were assessed. An ultimate 1% recovery rate of infectious viruses was achieved with the optimal protocol, corresponding to filtration through glass wool, followed by a viral elution step and then a PEG concentration. This method was validated for the detection of highly pathogenic H5N1 strains from artificially contaminated larger water volumes, from 10 to up to 50 liters, from different sources. The viral recovery efficiencies ranged from 0.01% to 7.89% and from 3.63% to 13.79% with lake water and rainwater, respectively. A theoretical detection threshold of 2.25 × 10
2
TCID
50
(50% tissue culture infectious dose) in the filtered volume was obtained for seeded lake waters by M gene reverse transcriptase PCR (RT-PCR). Moreover, the method was used successfully in field studies for the detection of naturally occurring influenza A viruses in lake water in France.
Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning
Authors: Ruecker Norma J. , Hoffman Rebecca M. , Chalmers Rachel M. , Neumann Norman F.
Published: April 2011

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of
Cryptosporidium
species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (
Cryptosporidium hominis
,
C. parvum
,
C. felis
,
C. meleagridis
,
C. ubiquitum
,
C. muris
, and
C. andersoni
) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for
C. andersoni
and
C. parvum
, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of
Cryptosporidium
at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of
Cryptosporidium
, particularly for environmental water samples, in which mixtures of parasites are common.
Unique Aliphatic Amidase from a Psychrotrophic and Haloalkaliphilic Nesterenkonia Isolate
Authors: Nel A. J. M. , Tuffin I. M. , Sewell B. T. , Cowan D. A.
Published: April 2011

Nesterenkonia
strain AN1 was isolated from a screening program for nitrile- and amide-hydrolyzing microorganisms in Antarctic desert soil samples. Strain AN1 showed significant 16S rRNA sequence identity to known members of the genus. Like known
Nesterenkonia
species, strain AN1 was obligately alkaliphilic (optimum environmental pH, 9 to 10) and halotolerant (optimum environmental Na
+
content, 0 to 15% [wt/vol]) but was also shown to be an obligate psychrophile with optimum growth at approximately 21°C. The partially sequenced genome of AN1 revealed an open reading frame (ORF) encoding a putative protein member of the nitrilase superfamily, referred to as NitN (264 amino acids). The protein crystallized readily as a dimer and the atomic structure of all but 10 amino acids of the protein was determined, confirming that the enzyme had an active site and a fold characteristic of the nitrilase superfamily. The protein was screened for activity against a variety of nitrile, carbamoyl, and amide substrates and was found to have only amidase activity. It had highest affinity for propionamide but demonstrated a low catalytic rate. NitN had maximal activity at 30°C and between pH 6.5 and 7.5, conditions which are outside the optimum growth range for the organism.
Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis
Authors: Lochner Adriane , Giannone Richard J. , Rodriguez Miguel , Shah Manesh B. , Mielenz Jonathan R. , Keller Martin , Antranikian Garabed , Graham David E. , Hettich Robert L.
Published: April 2011

The extremely thermophilic, Gram-positive bacteria
Caldicellulosiruptor bescii
and
Caldicellulosiruptor obsidiansis
efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from
C. bescii
and
C. obsidiansis
, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations:
C. obsidiansis
expressed the COB47_1671 protein with GH10/5 domains, while
C. bescii
expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The
C. obsidiansis
preparation, however, had higher cellulase specific activity and greater thermostability. The
C. bescii
culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.
GAP
Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris
Authors: Qin Xiulin , Qian Jiangchao , Yao Gaofeng , Zhuang Yingping , Zhang Siliang , Chu Ju
Published: April 2011

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast
Pichia pastoris
is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in
P. pastoris
, we created a functional promoter library through mutagenesis of the constitutive
GAP
promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and
S
-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in
P. pastoris
.
Biocontrol of Ralstonia solanacearum by Treatment with Lytic Bacteriophages
Authors: Fujiwara Akiko , Fujisawa Mariko , Hamasaki Ryosuke , Kawasaki Takeru , Fujie Makoto , Yamada Takashi
Published: April 2011

Ralstonia solanacearum
is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated
R. solanacearum
with three lytic phages: φRSA1, φRSB1, and φRSL1. Infection with φRSA1 and φRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with φRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with φRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All φRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. φRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active φRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as φRSL1, instead of a phage cocktail with highly virulent phages. Using this method, φRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting.
Killing of Candida albicans Filaments by Salmonella enterica Serovar Typhimurium Is Mediated by sopB Effectors, Parts of a Type III Secretion System
Authors: Kim Younghoon , Mylonakis Eleftherios
Published: April 2011

Although bacterial-fungal interactions shape microbial virulence during polymicrobial infections, only a limited number of studies have evaluated this interaction on a genetic level. We report here that one interaction is mediated by
sopB
, an effector of a type III secretion system (TTSS) of
Salmonella enterica
serovar Typhimurium. In these studies, we screened 10 TTSS effector-related mutants and determined their role in the killing of
C. albicans
filaments
in vitro
during coinfection in planktonic environments. We found that deleting the
sopB
gene (which encodes inositol phosphatase) was associated with a significant decrease in
C. albicans
killing at 25°C after 5 days, similar to that caused by the deletion of
sipB
(which encodes TTSS translocation machinery components). The
sopB
deletion dramatically influenced the killing of
C. albicans
filaments. It was associated with repressed filamentation in the
Caenorhabditis elegans
model of
C. albicans
-
S.
Typhimurium coinfection, as well as with biofilm formation by
C. albicans
. We confirmed that SopB translocated to fungal filaments through SipB during coinfection. Using quantitative real-time PCR assays, we found that the
Candida
supernatant upregulated the
S.
Typhimurium genes associated with
C. albicans
killing (
sopB
and
sipB
). Interestingly, the s
opB
effector negatively regulated the transcription of
CDC42
, which is involved in fungal viability. Taken together, these results indicate that specific TTSS effectors, including SopB, play a critical role in bacterial-fungal interactions and are important to
S.
Typhimurium in order to selectively compete with fungal pathogens. These findings highlight a new role for TTSS of
S.
Typhimurium in the intestinal tract and may further explain the evolution and maintenance of these traits.
The Paf1 Complex Represses
ARG1
Transcription in Saccharomyces cerevisiae by Promoting Histone Modifications
Authors: Crisucci Elia M. , Arndt Karen M.
Published: April 2011

The conserved multifunctional Paf1 complex is important for the proper transcription of numerous genes, and yet the exact mechanisms by which it controls gene expression remain unclear. While previous studies indicate that the Paf1 complex is a positive regulator of transcription, the repression of many genes also requires the Paf1 complex. In this study we used
ARG1
as a model gene to study transcriptional repression by the Paf1 complex in
Saccharomyces cerevisiae
. We found that several members of the Paf1 complex contribute to
ARG1
repression and that the complex localizes to the
ARG1
promoter and coding region in repressing conditions, which is consistent with a direct repressive function. Furthermore, Paf1 complex-dependent histone modifications are enriched at the
ARG1
locus in repressing conditions, and histone H3 lysine 4 methylation contributes to
ARG1
repression. Consistent with previous reports, histone H2B monoubiquitylation, the mark upstream of histone H3 lysine 4 methylation, is also important for
ARG1
repression. To begin to identify the mechanistic basis for Paf1 complex-mediated repression of
ARG1
, we focused on the Rtf1 subunit of the complex. Through an analysis of
RTF1
mutations that abrogate known Rtf1 activities, we found that Rtf1 mediates
ARG1
repression primarily by facilitating histone modifications. Other members of the Paf1 complex, such as Paf1, appear to repress
ARG1
through additional mechanisms. Together, our results suggest that Rtf1-dependent histone H2B ubiquitylation and H3 K4 methylation repress
ARG1
expression and that histone modifications normally associated with active transcription can occur at repressed loci and contribute to transcriptional repression.
Self-Induction of
a
/
a
or α/α Biofilms in Candida albicans Is a Pheromone-Based Paracrine System Requiring Switching
Authors: Yi Song , Sahni Nidhi , Daniels Karla J. , Lu Kevin L. , Huang Guanghua , Srikantha Thyagarajan , Soll David R.
Published: April 2011

Like
MTL
-heterozygous (
a
/α) cells, white
MTL
-homozygous (
a
/
a
or α/α) cells of
Candida albicans
, to which a minority of opaque cells of opposite mating type have been added, form thick, robust biofilms. The latter biofilms are uniquely stimulated by the pheromone released by opaque cells and are regulated by the mitogen-activated protein kinase signal transduction pathway. However, white
MTL
-homozygous cells, to which opaque cells of opposite mating type have not been added, form thinner biofilms. Mutant analyses reveal that these latter biofilms are self-induced. Self-induction of
a
/
a
biofilms requires expression of the α-receptor gene
STE2
and the α-pheromone gene
MF
α, and self-induction of α/α biofilms requires expression of the
a
-receptor gene
STE3
and the
a
-pheromone gene
MF
a
. In both cases, deletion of
WOR1
, the master switch gene, blocks cells in the white phenotype and biofilm formation, indicating that self-induction depends upon low frequency switching from the white to opaque phenotype. These results suggest a self-induction scenario in which minority opaque
a
/
a
cells formed by switching secrete, in a mating-type-nonspecific fashion, α-pheromone, which stimulates biofilm formation through activation of the α-pheromone receptor of majority white
a
/
a
cells. A similar scenario is suggested for a white α/α cell population, in which minority opaque α/α cells secrete
a
-pheromone. This represents a paracrine system in which one cell type (opaque) signals a second highly related cell type (white) to undergo a complex response, in this case the formation of a unisexual white cell biofilm.
Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough
Authors: da Silva Sofia M. , Pimentel Catarina , Valente Filipa M. A. , Rodrigues-Pousada Claudina , Pereira Inês A. C.
Published: April 2011

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of
Desulfovibrio vulgaris
Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC
3
protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC
3
specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.
Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen
Authors: Lai Tzung-Huei , Orellana Nelson G. , Yuasa Yumi , Rikihisa Yasuko
Published: April 2011

Anaplasma platysinfects peripheral blood platelets and causes infectious cyclic thrombocytopenia in canines. The genes, proteins, and antigens ofA. platysare largely unknown, and an antigen for serodiagnosis ofA. platyshas not yet been identified. In this study, we cloned theA. platysmajor outer membrane protein cluster, including the P44/Msp2 expression locus (p44ES/msp2ES) and outer membrane protein (OMP), using DNA isolated from the blood of four naturally infected dogs from Venezuela and Taiwan, Republic of China.A. platysp44ESis located within a 4-kb genomic region downstream from a putative transcriptional regulator,tr1, and a homolog of theAnaplasma phagocytophilum, identified here asA. platysomp-1X. The predicted molecular masses of the four matureA. platysP44ES proteins ranged from 43.3 to 43.5 kDa. Comparative analyses of the deduced amino acid sequences of Tr1, OMP-1X, and P44/Msp2 proteins fromA. platyswith those fromA. phagocytophilumshowed sequence identities of 86.4% for Tr1, 45.9% to 46.3% for OMP-1X, and 55.0% to 56.9% for P44/Msp2. Comparison betweenA. platysandAnaplasma marginaleproteins showed sequence identities of 73.1% for Tr1/Tr, 39.8% for OMP-1X/OMP1, and 41.5% to 42.1% for P44/Msp2. A synthetic OMP-1X peptide was shown to react withA. platys-positive sera but not withA. platys-negative sera orA. phagocytophilum-positive sera. Together, determination of the genomic locus ofA. platysouter membrane proteins not only contributes to the fundamental understanding of this enigmatic pathogen but also helps in developingA. platys-specific PCR and serodiagnosis.
Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329
Authors: Qu Yi , Spain Jim C.
Published: April 2011

Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by
Bradyrhizobium
sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds.
Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis
Authors: Tachon Sybille , Chambellon Emilie , Yvon Mireille
Published: April 2011

Water-forming NADH oxidases (encoded by
noxE
,
nox2
, or
nox
) are flavoproteins generally implicated in the aerobic survival of microaerophilic bacteria, such as lactic acid bacteria. However, some natural
Lactococcus lactis
strains produce an inactive NoxE. We examined the role of NoxE in the oxygen tolerance of
L. lactis
in the rich synthetic medium GM17. Inactivation of
noxE
suppressed 95% of NADH oxidase activity but only slightly affected aerobic growth, oxidative stress resistance, and NAD regeneration. However,
noxE
inactivation strongly impaired oxygen consumption and mixed-acid fermentation. We found that the A303T mutation is responsible for the loss of activity of a naturally occurring variant of NoxE. Replacement of A303 with T or G or of G307 with S or A by site-directed mutagenesis led to NoxE aggregation and the total loss of activity. We demonstrated that L299 is involved in NoxE activity, probably contributing to positioning flavin adenine dinucleotide (FAD) in the active site. These residues are part of the strongly conserved sequence LA(T)XXAXXXG included in an alpha helix that is present in other flavoprotein disulfide reductase (FDR) family flavoproteins that display very similar three-dimensional structures.
Lithium Diffusion in Li4/3Ti5/3O4 Particles during Insertion and Extraction
Authors: Takami Norio , Hoshina Keigo , Inagaki Hiroki
Published: April 2011

Lithium diffusion in a small Li4/3Ti5/3O4 (LTO) particle was investigated from kinetic viewpoints of two-phase transition process based on a core-shell model by means of galvanostatic and potentiostatic measurements of thin LTO composite electrodes. High-rate galvanostatic charge (insertion) – discharge (extraction) properties of the thin LTO composite electrode showed that the insertion into the LTO particle was significantly slower than the extraction. An apparent chemical diffusion coefficient (Dapp
) of lithium in the LTO particle during the insertion and extraction was evaluated from the results of potential step chronoamperometry (PSCA) with a spherical finite diffusion model. The phase-boundary movements between the two phases in the cathodic and the anodic potential steps for a long-time region were controlled by lithium diffusion through Li7/3Ti5/3O4 rock-salt (LTO-rock-salt) and Li4/3Ti5/3O4 spinel (LTO-spinel) shell, respectively. Dapp
in the LTO-rock-salt and the LTO-spinel phase were estimated to be approximately 1 × 10−12 cm2/s and 1.6 × 10−11 cm2/s, respectively. The slower insertion was mainly due to a Dapp
value one order of magnitude smaller in the LTO-rock-salt than that in the LTO-spinel phase. Electrochemical kinetic properties of the LTO particle with the core-shell structure were interpreted by lithium diffusion through the LTO-rock-salt shell during the insertion and the LTO-spinel shell with the low electron conductivity during the extraction.
Load Transients of Nanostructured Thin Film Electrodes in Polymer Electrolyte Fuel Cells
Authors: Kongkanand Anusorn , Sinha Puneet K.
Published: April 2011

Transient responses of conventional carbon-supported Pt nanoparticle (Pt/C) and 3M’s nano structured thin film (NSTF) oxygen reduction electrodes are reported. The ultrathin 0.3 μm thick NSTF electrode exhibits less-than-zero voltage, i.e. a failed up-transient, when current is increased instantaneously from 0.02 to 1.0 A/cm2 at 80°C and the channel RH is greater than 80%. In contrast, under the same conditions, a 10 μm thick Pt/C electrode exhibits robust transient response. A transient model, coupled with experiments, is deployed to delineate the water transport dynamics and ensuing voltage response. It is found that thin electrodes, due to their low water storage capacities, flood with liquid water rendering negative cell voltages during transients. Effects of operating conditions and material properties such as membrane and electrode thickness on the transient response are investigated.
An Overview of Patterned Metal/Dielectric Surface Bonding: Mechanism, Alignment and Characterization
Authors: Di Cioccio L. , Gueguen P. , Taibi R. , Landru D. , Gaudin G. , Chappaz C. , Rieutord F. , de Crecy F. , Radu I. , Chapelon L. L. , Clavelier L.
Published: April 2011

An overview of the different metal bonding techniques used for 3D integration is presented. Key parameters such as surface preparation, temperature and duration of annealing, achievable wafer-to-wafer alignment and electrical results are reviewed. A special focus is done on direct bonding of patterned metal/dielectric surfaces. A mechanism for copper direct bonding is proposed based on bonding toughness measurements, SAM, XRR, XRD, and TEM analysis. Dedicated characterization techniques for such bonding are presented.
Investigation of the Irreversible Capacity Loss in the Lithium-Rich Oxide Li[Li1/5Ni1/5Mn3/5]O2
Authors: van Bommel Andrew , Krause L. J. , Dahn J. R.
Published: April 2011

The lithium-rich transition metal oxides show a larger first charge capacity and larger cycling capacities than the non-lithium-rich transition metal oxides. The disadvantages of the lithium-rich transition metal oxides include relatively poor rate capabilities and relatively large irreversible capacities. In this report, the irreversible capacity loss of the lithium rich oxide Li[Li1/5Ni1/5Mn3/5]O2 was investigated. Stepwise traverse of the oxygen-release plateau increased the cycling capacity of Li/Li[Li1/5Ni1/5Mn3/5]O2 cells and gave evidence that lithium was removed from the transition metal layer at the start of the oxygen release plateau. The irreversible capacity loss was attributed to the diffusion of transition metals into the lithium vacancies in the transition metal layer and the subsequent inability for lithium reinsertion into the transition metal layer. Isothermal calorimetry of Li/Li[Li1/5Ni1/5Mn3/5]O2 cells cycled from 2.5 to 4.4 V (no oxygen loss) supported the view that lithium is not deintercalated from the transition metal layer at the start of charge.
Regulator of G protein signaling 2 is a functionally important negative regulator of angiotensin II-induced cardiac fibroblast responses
Authors: Zhang Peng , Su Jialin , King Michelle E. , Maldonado Angel E. , Park Cindy , Mende Ulrike
Published: April 2011

Cardiac fibroblasts play a key role in fibrosis development in response to stress and injury. Angiotensin II (ANG II) is a major profibrotic activator whose downstream effects (such as phospholipase Cβ activation, cell proliferation, and extracellular matrix secretion) are mainly mediated via Gq-coupled AT1 receptors. Regulators of G protein signaling (RGS), which accelerate termination of G protein signaling, are expressed in the myocardium. Among them, RGS2 has emerged as an important player in modulating Gq-mediated hypertrophic remodeling in cardiac myocytes. To date, no information is available on RGS in cardiac fibroblasts. We tested the hypothesis that RGS2 is an important regulator of ANG II-induced signaling and function in ventricular fibroblasts. Using an in vitro model of fibroblast activation, we have demonstrated expression of several RGS isoforms, among which only RGS2 was transiently upregulated after short-term ANG II stimulation. Similar results were obtained in fibroblasts isolated from rat hearts after in vivo ANG II infusion via minipumps for 1 day. In contrast, prolonged ANG II stimulation (3–14 days) markedly downregulated RGS2 in vivo. To delineate the functional effects of RGS expression changes, we used gain- and loss-of-function approaches. Adenovirally infected RGS2 had a negative regulatory effect on ANG II-induced phospholipase Cβ activity, cell proliferation, and total collagen production, whereas RNA interference of endogenous RGS2 had opposite effects, despite the presence of several other RGS. Together, these data suggest that RGS2 is a functionally important negative regulator of ANG II-induced cardiac fibroblast responses that may play a role in ANG II-induced fibrosis development.