bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces
Authors: Peterson Robyn , Grinyer Jasmine , Nevalainen Helena 
Published: April 2011

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.