bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes byZymomonas mobilis
Authors: Linger Jeffrey G. , Adney William S. , Darzins Al 
Published: August 2010

ABSTRACTDevelopment of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation hostZymomonas mobiliswere investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains ofZ. mobiliswere found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 fromAcidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes inZ. mobilisalthough to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native toZ. mobilisto the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest thatZ. mobilisis capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developingZ. mobilisinto a CBP platform organism.
Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain
Authors: Wang Hai-Hong , Riding Stephen , Lindo Paul , Singh Bal Ram 
Published: August 2010

ABSTRACT

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by
Clostridium botulinum
, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.
Evaluating the Growth Potential of Pathogenic Bacteria in Water
Authors: Vital Marius , Stucki David , Egli Thomas , Hammes Frederik 
Published: August 2010

ABSTRACT

The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (
Escherichia coli
O157,
Vibrio cholerae
, or
Pseudomonas aeruginosa
) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.
Influence of the Diversity of Bacterial Isolates from Drinking Water on Resistance of Biofilms to Disinfection
Authors: Simões Lúcia Chaves , Simões Manuel , Vieira Maria João 
Published: August 2010

ABSTRACT

Single- and multispecies biofilms formed by six drinking water-isolated bacterial species were used to assess their susceptibilities to sodium hypochlorite (SHC). In general, multispecies biofilms were more resistant to inactivation and removal than single biofilms. Total biofilm inactivation was achieved only for
Acinetobacter calcoaceticus
single-species biofilms and for those multispecies biofilms without
A. calcoaceticus
. Biofilms with all bacteria had the highest resistance to SHC, while those without
A. calcoaceticus
were the most susceptible.
A. calcoaceticus
formed single biofilms susceptible to SHC; however, its presence in multispecies biofilms increased their resistance to disinfection.
Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in
Saccharomyces cerevisiae
Authors: Kitajima Toshihiko , Chiba Yasunori , Jigami Yoshifumi 
Published: August 2010

ABSTRACT

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast
Pichia pastoris
that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of
Saccharomyces cerevisiae
and the identification of a mutant allele of the
MUP1
gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the
mup1
mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the
mup1
mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the
mup1
mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.
Anaerobic Biotransformation of High Concentrations of Chloroform by an Enrichment Culture and Two Bacterial Isolates
Authors: Shan Huifeng , Kurtz Harry D. , Mykytczuk Nadia , Trevors Jack T. , Freedman David L. 
Published: August 2010

ABSTRACT

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two
Pantoea
spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B
12
(approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO
2
, and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.
Development of
pyrF-
Based Genetic System for Targeted Gene Deletion in
Clostridium thermocellum
and Creation of a
pta
Mutant
Authors: Tripathi Shital A. , Olson Daniel G. , Argyros D. Aaron , Miller Bethany B. , Barrett Trisha F. , Murphy Daniel M. , McCool Jesse D. , Warner Anne K. , Rajgarhia Vineet B. , Lynd Lee R. , Hogsett David A. , Caiazza Nicky C. 
Published: August 2010

ABSTRACT

We report development of a genetic system for making targeted gene knockouts in
Clostridium thermocellum
, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the
pyrF
gene. The Δ
pyrF
strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of
pyrF
from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed
pyrF
, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely
pta
, which encodes the enzyme phosphotransacetylase. The
C. thermocellum
Δ
pta
strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in
C. thermocellum
, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.
Quantification of Enteric Viruses, Pathogen Indicators, and
Salmonella
Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods
Authors: Wong Kelvin , Onan Brandon M. , Xagoraraki Irene 
Published: August 2010

ABSTRACT

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of
Salmonella enterica
in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10
4
MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for
Salmonella enterica
, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.
The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton
Authors: Luginbuehl Edith , Ryter Damaris , Schranz-Zumkehr Judith , Oberholzer Michael , Kunz Stefan , Seebeck Thomas 
Published: August 2010

ABSTRACT

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen
Trypanosoma brucei
, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes,
T. brucei
PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.
Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans
Authors: Hervás-Aguilar América , Peñalva Miguel A. 
Published: August 2010

ABSTRACTTheAspergillus nidulansendocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressibleslaB1allele for conditional SlaB expression. Video microscopy showed that repressedslaB1cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressedslaB1germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulatedslaB1cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.
Conserved Motifs Involved in ATP Hydrolysis by MalT, a Signal Transduction ATPase with Numerous Domains fromEscherichia coli
Authors: Marquenet Emélie , Richet Evelyne 
Published: August 2010

ABSTRACTThe signal transduction ATPases with numerous domains (STAND) are sophisticated signaling proteins that are related to AAA+proteins and control various biological processes, including apoptosis, gene expression, and innate immunity. They function as tightly regulated switches, with the off and on positions corresponding to an ADP-bound, monomeric form and an ATP-bound, multimeric form, respectively. Protein activation is triggered by inducer binding to the sensor domain. ATP hydrolysis by the nucleotide-binding oligomerization domain (NOD) ensures the generation of the ADP-bound form. Here, we use MalT, anEscherichia colitranscription activator, as a model system to identify STAND conserved motifs involved in ATP hydrolysis besides the catalytic acidic residue. Alanine substitution of the conserved polar residue (H131) that is located two residues downstream from the catalytic residue (D129) blocks ATP hydrolysis and traps MalT in an active, ATP-bound, multimeric form. This polar residue is also conserved in AAA+. Based on AAA+X-ray structures, we proposed that it is responsible for the proper positioning of the catalytic and the sensor I residues for the hydrolytic attack. Alanine substitution of the putative STAND sensor I (R160) abolished MalT activity. Substitutions of R171 impaired both ATP hydrolysis and multimerization, which is consistent with an arginine finger function and provides further evidence that ATP hydrolysis is primarily catalyzed by MalT multimers.
Actin-Based Motility of
Burkholderia thailandensis
Requires a Central Acidic Domain of BimA That Recruits and Activates the Cellular Arp2/3 Complex
Authors: Sitthidet Chayada , Stevens Joanne M. , Field Terence R. , Layton Abigail N. , Korbsrisate Sunee , Stevens Mark P. 
Published: August 2010

ABSTRACT

Burkholderia
species use BimA for intracellular actin-based motility. Uniquely,
Burkholderia thailandensis
BimA harbors a central and acidic (CA) domain. The CA domain was required for actin-based motility, binding to the cellular Arp2/3 complex, and Arp2/3-dependent polymerization of actin monomers. Our data reveal distinct strategies for actin-based motility among
Burkholderia
species.
Personalized Medicine: Marking a New Epoch in Cancer Patient Management
Authors: Diamandis Maria , White Nicole M.A. , Yousef George M. 
Published: August 2010

AbstractPersonalized medicine (PM) is defined as “a form of medicine that uses information about a person's genes, proteins, and environment to prevent, diagnose, and treat disease.” The promise of PM has been on us for years. The suite of clinical applications of PM in cancer is broad, encompassing screening, diagnosis, prognosis, prediction of treatment efficacy, patient follow-up after surgery for early detection of recurrence, and the stratification of patients into cancer subgroup categories, allowing for individualized therapy. PM aims to eliminate the “one size fits all” model of medicine, which has centered on reaction to disease based on average responses to care. By dividing patients into unique cancer subgroups, treatment and follow-up can be tailored for each individual according to disease aggressiveness and the ability to respond to a certain treatment. PM is also shifting the emphasis of patient management from primary patient care to prevention and early intervention for high-risk individuals. In addition to classic single molecular markers, high-throughput approaches can be used for PM including whole genome sequencing, single-nucleotide polymorphism analysis, microarray analysis, and mass spectrometry. A common trend among these tools is their ability to analyze many targets simultaneously, thus increasing the sensitivity, specificity, and accuracy of biomarker discovery. Certain challenges need to be addressed in our transition to PM including assessment of cost, test standardization, and ethical issues. It is clear that PM will gradually continue to be incorporated into cancer patient management and will have a significant impact on our health care in the future. Mol Cancer Res; 8(9); 1175–87. ©2010 AACR.
CLOSE-IN FALLOUT
Authors: Kellogg W. W. , Rapp R. R. , Greenfield S. M. 
Published: August 2010

Abstract
The phenomenon of radioactive fallout from an atomic explosion is described, and a quantitative technique for determining the distribution of radioactive material on the ground is developed. The primary factors which must be considered are wind field, yield and height of burst, and particle-size distribution. Certain parameters which enter directly into a fallout determination are given quantitatively, such as the altitude and size of the atomic cloud (as a function of explosion yield and atmospheric stability) and particle fall-rates (as a function of altitude and particle size). Two hypothetical fallout patterns for a one-megaton explosion, computed on a high-speed digital computer are presented, showing the large effect which the wind has in determining the character of the fallout. The meteorological problems associated with a fallout prediction are discussed.
A STUDY OF THE RAINFALL PATTERNS AND SOME RELATED FEATURES IN A DISSIPATING HURRICANE
Authors: Gilbert Sterling C. , LaSeur N. E. 
Published: August 2010

Abstract
A detailed investigation of the hourly rainfall patterns accompanying a dissipating hurricane over the southeastern United States demonstrates the pronounced asymmetry in the decay of release of latent heat, the primary energy source of the storm. This appears to be associated with the incorporation into the storm circulation of dry air in the layer from 800 to 400 millibars. This air is first drawn in from the outer portions of the left-front quadrant, then sweeps rapidly around thestorm into the right-rearand right-front quadrants.
ON THE STANDARD VECTOR-DEVIATION WIND ROSE
Authors: Crutcher Harold L. 
Published: August 2010

Abstract
Wind-data presentations are varied. Some serve one purpose better than another. A relatively new type of wind rose is presented in order that its potential may be exploited. This is the standard vector-deviation wind rose. It is an easily reproduced rose and may be constructed from values of the vector mean wind, standard deviations of the latitudinal and meridional wind components, and the correlation between the components. If the distribution is circular, rather than elliptical, only the vector mean and the standard vector deviation are needed. Methods to test for ellipticity and to construct the roses are given.