bluuebunny/crossref_metadata_2025 huggingface dataset:
Air Dispersion Model to Forecast Pollutant Concentration Around Thermal Power Plants
Authors: Huertas Jose´ I. , Carmona Mauricio Y. , Moreno Diego
Published: October 2008

The Mexican environmental authority requires that thermal power plants operate 3 or 4 air quality monitoring stations around its main stack to ensure that pollutant concentration levels are always below the maximum allowable. However the high cost of these stations and the cost of their maintenance have made this regulation economically unreasonable. It has been proposed to reduce the number of monitoring stations to one and substitute the other stations by an accurate atmospheric dispersion model that allows the permanent surveillance of the surface pollutant concentration levels around the thermoelectric power plants. CALPUFF, an advanced air pollution dispersion modeling system was implemented for the special case of the Mexican thermal power plants. Experimental work was conducted to verify the correct implementation of the model. This paper describes the main results obtained during the development of this work.
Verification and Validation in Computational Fluid Dynamics and Heat Transfer: PTC 61
Authors: Freitas Christopher J.
Published: October 2008

ASME Codes and Standards created a new Performance Test Code Committee in May of 2004, whose primary focus is on the formalization of an approach to the process for verification and validation in computational fluid dynamics and heat transfer. PTC-61 has as its charter to provide “procedures for quantifying the accuracy of modeling and simulation in computational fluid dynamics and heat transfer.” The committee is initially focusing on an approach based on the concepts and methods of experimental uncertainty analysis. This will leverage the decades of development in the community of experimentalists that resulted in the ASME Standard PTC 19.1 “Test Uncertainty” and the ISO international standard “Guide to the Expression of Uncertainty in Measurement.” This paper provides a brief summary of the status of the PTC-61 effort to develop a standard for V&V.
Evaluation of the Last Stage Blades of 37.5 and 110 MW Geothermal Turbines
Authors: Kubiak J. , Sierra F. , Garci´a J. C. , Gonza´lez J. G. , Nebradt J. , Urquiza G.
Published: October 2008

This paper presents the results of the evaluation and investigation of the last stage blades of 37.5 and 110 MW geothermal turbines. In the investigation, the operational data and the history of the blade failures were analysed for several rotors of the units. The results of previous repairs were reviewed, and metallurgical investigations conducted. The mechanical and metallurgical modes of failures of some of the fractured blades were identified. A finite element analysis (FEA) of the blades was carried out to calculate the natural frequencies and the vibratory stresses on the blades, which were used to estimate their design useful life. Then the natural frequencies of the installed blades were measured in both, on the rotor and in the laboratory. It was observed that deterioration of the blades was caused in general by processes resulting from moisture condensation in the steam: erosion and corrosion. Also, the results of the investigation showed that the fractures of the two kinds of blades were attributed to the errors during the installation and manufacturing. Additionally, general deterioration of the blades contributed to the fracture. The paper discusses the way of this contribution. It also addresses what kind of recommendations were issued to amend the problem, whether repair or replace using the original or new design blades.
DLN Technology Update for the M701DA: Enhancing Vintage Gas Turbine Models With Latest Technology
Authors: Tanimura Satoshi , Komori Toyoaki , Fukuizumi Yasushi , Cloyd Scott , McDeed David
Published: October 2008

While the Gas Turbine design process is often focused on the development of the next advanced frame significant benefits can be achieved by retrofitting the latest technology in mature gas turbines model. This dissemination of advanced technology is a company philosophy of constant equipment enhancement. This paper will track the design process that is typically followed when enhancing vintage equipment. Also consideration will be discussed of transferring technology between 50 and 60 Hert frequency fleets.
Benchmarking Software for Slagging, Fouling and Other Parameters to Improve Coal-Fired Power Plant Load Factor, Efficiency and Emission
Authors: Parker Ken R. , Allen Jeff , Sanyal Anupam
Published: October 2008

For a 500 MW unit, a 1 % reduction in boiler efficiency equates to a coal cost in the order of $ 1/2 million/annum, while a 5 % unit derate, needed to meet emission compliance, can equate to an annual revenue loss of up to $ 12 million. Many software products are in use, which based on the present plant performance, identify and optimize the operational parameters for the best plant capacity, utilization and efficiency. While they are effective in tuning up the unit, they are applied to an operating plant firing a given coal for the given performance. But what if there were a means of predicting how a coal will perform with respect to slagging, fouling and every other single parameter involved in the use of that coal for capacity, efficiency maximization and emission control without firing even a lb of coal? This would enable the operator to know in advance what to expect and thereby, adjust the operating variables to get the best out of that coal. Such a software product – SCES (Steam Coal Evaluation & Services) has been developed based on the fundamental principles of combustion, mineral matter transformation and emission of particulates, NOx, SO2 and mercury, based on only the standard ASTM coal and ash analyses. The operational parameters evaluated are: Slagging and fouling as well as Grindability, Abrasion of the grinding elements; Combustibility & Unburnt carbon, Corrosion and Erosion; Emission of particulates, the oxides of sulfur and nitrogen (both primary and secondary DeNOx) as well as mercury when data are available. Its advantage over the existing products is its ability to predict, amongst others parameters, corrosion, erosion of convective tubes and the life of grinding elements none of which are discernible from the existing software products or from a limited test burn. These parameters however play important roles in the bottom line O & M costs. The implementation of SCES in providing meaningful rankings alerts the plant operators to the performance they can expect and any measures that need to be taken to be able the plant to operate at its highest load factor and efficiency while under emission compliance with minimum impact on O & M cost. No separate coal sample or small-scale laboratory evaluation work is required in the derivation of the rankings. Because of its simplicity of use and immediate availability of results, SCES can also be used as a routine analytical tool, accompanying the coal analyses by the plant or supplied with each delivery of coal. It is applicable to all coals irrespective of rank and country of origin, it has been used and validated on coals from the US, UK, Russia, Columbia and India. The paper describes the fundamental properties coal used in the development of the software and cites case histories of its validation on US (Bituminous & Sub-bituminous) coals.
Water Quality Controls in Construction
Authors: Wookey Claude J.
Published: October 2008

Water Quality control for construction sites is a requirement at any site within the United States. Erosion Controls are the first, and many times, last preventative measure for water discharge from a site. If these fail, streams, wetlands, or even off-site upland areas (yards, etc.) are adversely affected. Site quality is affected when valuable soil is lost and restoration requires replacement. Human exposure to contaminants, violation of governmental regulations, contractor reputation, and the overall economics involved in construction are at risk.
Variations inIxodes ricinusDensity andBorreliaInfections Associated with Cattle Introduced into a Woodland in The Netherlands
Authors: Gassner Fedor , Verbaarschot Patrick , Smallegange Renate C. , Spitzen Jeroen , Van Wieren Sipke E. , Takken Willem
Published: October 2008

The effect of introduced large herbivores on the abundance ofIxodes ricinusticks and theirBorreliainfections was studied in a natural woodland in The Netherlands. Oak and pine plots, either ungrazed or grazed by cattle, were selected. Ticks were collected weekly by blanket dragging.Borreliainfections were determined by PCR and restriction fragment length polymorphism. Rodent densities were estimated using mark-release-recapture methods. On occasion, the cattle were inspected for tick infestations. Meteorological data were recorded for each habitat. Significantly more ticks were collected in the ungrazed woodland than in the grazed woodland. The ungrazed oak habitat had higher tick densities than the pine habitat, while in the grazed habitats, tick densities were similar.Borreliainfection rates ranged from zero in larvae to 26% in nymphs to 33% in adult ticks, andB. afzelii,B. burgdorferisensu stricto,B. garinii, andB. valaisianawere the species involved. Coinfections were found in five ticks. There was no effect of the presence of cattle onBorreliainfections in the ticks. In the ungrazed area,Borreliainfections in nymphs were significantly higher in the oak habitat than in the pine habitat. More mice were captured in the ungrazed area, and these had a significantly higher tick burden than mice from the grazed area. Tick burden on cattle was low. The results suggest that grazing has a negative effect on small rodents as well as on ticks but not onBorreliainfections. Implications of these results for management of woodland reserves and risk of Lyme disease are discussed.
Comparative Analysis of Extracellular and Intracellular Proteomes ofListeria monocytogenesStrains Reveals a Correlation between Protein Expression and Serovar
Authors: Dumas Emilie , Meunier Bruno , Berdagué Jean-Louis , Chambon Christophe , Desvaux Mickaël , Hébraud Michel
Published: October 2008

Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity ofL. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with theL. monocytogenesphylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.
HtrA Is Essential for Efficient Secretion of Recombinant Proteins by
Lactococcus lactis
Authors: Sriraman Kalpana , Jayaraman Guhan
Published: October 2008

HtrA is a unique protease on the extracellular surface of
Lactococcus lactis
. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of
htrA
can lead to the complete stabilization of recombinant proteins. In this work, we have shown that
htrA
mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in
L. lactis
. Instead of using an
L. lactis htrA
mutant, the reduction of the HtrA level in wild-type recombinant cultures of
L. lactis
by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.
Mechanism and Consequences of Anaerobic Respiration of Cobalt byShewanella oneidensisStrain MR-1
Authors: Hau Heidi H. , Gilbert Alan , Coursolle Dan , Gralnick Jeffrey A.
Published: October 2008

Bacteria from the genusShewanellaare the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability ofShewanella oneidensisto catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA]−reduction byS. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA]2−, the product of [Co(III)-EDTA]−respiration, inhibited the growth ofS. oneidensisstrain MR-1 and that this toxicity was partially abolished by the addition of MgSO4. We demonstrate that [Co(III)-EDTA]−reduction byS. oneidensisrequires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (thec-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developingS. oneidensisstrains as a tool for bioremediation.
Characterization of a Second
Rhodococcus erythropolis
SQ1 3-Ketosteroid 9α-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB
Authors: van der Geize R. , Hessels G. I. , Nienhuis-Kuiper M. , Dijkhuizen L.
Published: October 2008

Previously we have characterized 3-ketosteroid 9α-hydroxylase (KSH), a key enzyme in microbial steroid degradation in
Rhodococcus erythropolis
strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the
kshA1
gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the
kshA2
gene in mutant strain RG2 restored growth on AD and ADD, indicating that
kshA2
also encodes KSH activity. The functional complementation was shown to be dependent on the presence of
kshB
. Transcriptional analysis showed that expression of
kshA2
is induced in parent strain
R. erythropolis
SQ1 in the presence of AD. However, promoter activity studies, using β-lactamase of
Escherichia coli
as a convenient transcription reporter protein for
Rhodococcus
, revealed that the
kshA2
promoter in fact is highly induced in the presence of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of
kshA2
in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third
kshA
homologue was additionally identified in a
kshA1 kshA2
double gene deletion mutant strain of
R. erythropolis
SQ1. The developed degenerate PCR primers for
kshA
may be useful for isolation of
kshA
homologues from other (actino) bacteria.
SOD1, a New
Kluyveromyces lactis
Helper Gene for Heterologous Protein Secretion
Authors: Raimondi S. , Zanni E. , Talora C. , Rossi M. , Palleschi C. , Uccelletti D.
Published: October 2008

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast.
Saccharomyces cerevisiae
cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses.
SOD1
encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O
2
−
) into oxygen and hydrogen peroxide. It is a Cu
2+
/Zn
2+
metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the
Kluyveromyces lactis SOD1
(Kl
SOD1
) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from
Arxula adeninivorans
. In addition, Kl
SOD1
overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when
K. lactis
cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing Kl
SOD1
showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.
Cloning and Heterologous Expression of Insecticidal-Protein-Encoding Genes from
Photorhabdus luminescens
TT01 in
Enterobacter cloacae
for Termite Control
Authors: Zhao Ruihua , Han Richou , Qiu Xuehong , Yan Xun , Cao Li , Liu Xiuling
Published: October 2008

Enterobacter cloacae
, one of the indigenous gut bacteria of the Formosan subterranean termite (
Coptotermes formosanus
), was genetically modified with a transposon Tn
5
vector containing genes (
tcdA1
and
tcdB1
) encoding orally insecticidal proteins from the entomopathogenic bacterium
Photorhabdus luminescens
subsp.
laumondii
TT01, a symbiont of the entomopathogenic nematode
Heterorhabditis bacteriophora
, for termite control. In the laboratory, termites were fed filter paper inoculated with the recombinant bacteria. The chromosomal expression of the introduced genes showed that there were insecticidal activities against termite workers and soldiers challenged with the transformed bacteria. After termites were fed recombinant bacteria, the termite mortality was 3.3% at day 5, and it increased from 8.7% at day 9 to 93.3% at day 29. All the dead termites contained the recombinant bacteria in their guts. Transfer of the recombinant bacteria occurred between donor workers (initially fed recombinant bacteria) and recipient workers (not fed). More than 20% of the recipient termites ingested recombinant bacteria within 2 h, and 73.3% of them had ingested recombinant bacteria after 12 h. The method described here provides a useful alternative for sustainable control of the Formosan subterranean termite (
C. formosanus
) and other social insects, such as the imported red fire ant (
Solenopsis invicta
).
Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes
Authors: Duan Faping , Curtis Katherine L. , March John C.
Published: October 2008

Here, we show that commensal bacteria can stimulate intestinal epithelial cells to secrete insulin in response to glucose. Commensal strains were engineered to secrete the insulinotropic proteins GLP-1 and PDX-1. Epithelia stimulated by engineered strains and glucose secreted up to 1 ng ml
−1
of insulin with no significant background secretion.
Host Adaptation of aWolbachiaStrain after Long-Term Serial Passage in Mosquito Cell Lines
Authors: McMeniman Conor J. , Lane Amanda M. , Fong Amy W. C. , Voronin Denis A. , Iturbe-Ormaetxe Iñaki , Yamada Ryuichi , McGraw Elizabeth A. , O'Neill Scott L.
Published: October 2008

The horizontal transfer of the bacteriumWolbachia pipientisbetween invertebrate hosts hinges on the ability ofWolbachiato adapt to new intracellular environments. The experimental transfer ofWolbachiabetween distantly related host species often results in the loss of infection, presumably due to an inability ofWolbachiato adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shorteningWolbachiastrain,wMelPop, from the fruit flyDrosophila melanogasterinto a cell line derived from the mosquitoAedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adaptedwMelPop into cell lines derived from two other mosquito species,Aedes aegyptiandAnopheles gambiae. After a prolonged period of serial passage in mosquito cell lines,wMelPop was reintroduced into its native host,D. melanogaster, by embryonic microinjection. The cell line-adaptedwMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadaptWolbachiato novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.
Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in theListeria monocytogenesVirulence-Associated GeneinlA
Authors: Van Stelten A. , Nightingale K. K.
Published: October 2008

The virulence factor internalin A (InlA) facilitates the uptake ofListeria monocytogenesby epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations ininlAand demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700L. monocytogenesisolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing ofinlAgenes from isolates with attenuated invasion capacities revealed three novelinlAPMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and withoutinlAPMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations ininlA. The SNP genotyping assay detects allinlAPMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novelinlAPMSC mutations. PMSCs ininlAwere significantly (ribotypes DUP-1039C and DUP-1045B;P< 0.001) or marginally (ribotype DUP-1062D;P= 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S.L. monocytogenesisolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiateL. monocytogenesisolates carrying virulence-attenuating PMSC mutations ininlAfrom strains representing the most significant health risk.
A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by
Listeria monocytogenes
Authors: Zhu Xinna , Long Fei , Chen Yonghui , Knøchel Susanne , She Qunxin , Shi Xianming
Published: October 2008

Listeria monocytogenes
may persist for long periods in food processing environments. In some instances, this may be due to aggregation or biofilm formation. To investigate the mechanism controlling biofilm formation in the food-borne pathogen
L. monocytogenes
, we characterized LM-49, a mutant with enhanced ability of biofilm formation generated via transposon Tn
917
mutagenesis of
L. monocytogenes
4b G. In this mutant, a Tn
917
insertion has disrupted the coding region of the gene encoding a putative ATP-binding cassette (ABC) transporter permease identical to Lmof2365_1771 (a putative ABC transporter permease) presented in the sequenced strain
L. monocytogenes
strain 4b F2365. This disrupted gene, denoted lm.G_1771, encoded a protein with 10 transmembrane helixes. The revertant, LM-49RE, was obtained by replacing lm.G_1771::
Tn917
with lm.G_1771 via homologous recombination. We found that LM-49RE formed the same amount of biofilm biomass as the wild-type strain. Furthermore, transcription of the downstream lm.G_1770 gene was not influenced by the upstream Tn
917
insertion, and the presence of Tn
917
has no effect on biofilm formation. These results suggest that lm.G_1771 was solely responsible for the negative regulation of biofilm formation by
L. monocytogenes
4b G. The immediate gene upstream of lm.G_1771 encoded an ATP-binding protein. Bioinformatics analysis suggested that these two genes were organized into an operon and that their proteins formed an export ABC transporter. Here, we report the characterization of the mutant and identification of a novel ABC transporter that functions in negative regulation of biofilm formation in
L. monocytogenes
.
Persistent Association of
Mycobacterium ulcerans
with West African Predaceous Insects of the Family Belostomatidae
Authors: Mosi Lydia , Williamson Heather , Wallace John R. , Merritt Richard W. , Small P. L. C.
Published: October 2008

A number of studies have suggested that
Mycobacterium ulcerans
, the etiological agent of Buruli ulcer, may be transmitted to humans by insect bites.
M. ulcerans
has been isolated from a predaceous aquatic insect, and PCR detection of
M. ulcerans
DNA in aquatic environments suggests that the organism is widely distributed within many invertebrate taxa and functional feeding groups. Thus,
M. ulcerans
may be concentrated through different trophic links. However, the specific environmental niche of
M. ulcerans
and route of transmission to humans remain a mystery. In this study, a biologically relevant infection model in which
M. ulcerans
-infected mosquito larvae were fed to a species of predaceous hemiptera (African Belostomatidae) was used to demonstrate the persistent colonization of
M. ulcerans
and subsequent transmission of bacteria to naïve prey. The association of
M. ulcerans
with specific anatomical compartments showed that
M. ulcerans
accumulates preferentially on the exoskeleton. In contrast, few organisms were found in dissected guts or salivary glands. No difference was found between the ability of wild-type
M. ulcerans
and an
M. ulcerans
isogenic mycolactone-negative mutant to colonize belostomatids. These data show that African belostomatids can successfully be colonized by
M. ulcerans
and support the trophic transfer of
M. ulcerans
within the environment.
Expression Profiles and Physiological Roles of Two Types of Molecular Chaperonins from the Hyperthermophilic Archaeon
Thermococcus kodakarensis
Authors: Fujiwara Shinsuke , Aki Ryohei , Yoshida Masaya , Higashibata Hiroki , Imanaka Tadayuki , Fukuda Wakao
Published: October 2008

Thermococcus kodakarensis
possesses two chaperonins, CpkA and CpkB, and their expression is induced by the downshift and upshift, respectively, of the cell cultivation temperature. The expression levels of the chaperonins were examined by using specific antibodies at various cell growth temperatures in the logarithmic and stationary phases. At 60°C, CpkA was highly expressed in both the logarithmic and stationary phases; however, CpkB was not expressed in either phase. At 85°C, CpkA and CpkB were expressed in both phases; however, the CpkA level was decreased in the stationary phase. At 93°C, CpkA was expressed only in the logarithmic phase and not in the stationary phase. In contrast, CpkB was highly expressed in both phases. The results of reverse transcription-PCR experiments showed the same growth phase- and temperature-dependent profiles as observed in immunoblot analyses, indicating that the expression of
cpkA
and
cpkB
is regulated at the mRNA level. The
cpkA
or
cpkB
gene disruptant was then constructed, and its growth profile was monitored. The
cpkA
disruptant showed poor cell growth at 60°C but no significant defects at 85°C and 93°C. On the other hand,
cpkB
disruption led to growth defects at 93°C but no significant defects at 60°C and 85°C. These data indicate that CpkA and CpkB are necessary for cell growth at lower and higher temperatures, respectively. The logarithmic-phase-dependent expression of CpkA at 93°C suggested that CpkA participates in initial cell growth in addition to lower-temperature adaptation. Promoter mapping and quantitative analyses using the Phr (
Pyrococcus
heat-shock regulator) gene disruptant revealed that temperature-dependent expression was achieved in a Phr-independent manner.
Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products ofVibrio splendidus
Authors: Binesse Johan , Delsert Claude , Saulnier Denis , Champomier-Vergès Marie-Christine , Zagorec Monique , Munier-Lehmann Hélène , Mazel Didier , Le Roux Frédérique
Published: October 2008

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors ofVibriospecies; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogenVibrio splendidusLGP32 and show that toxicity is correlated to the presence of a metalloprotease and its correspondingvsmgene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and avsmdeletion mutant (Δvsmmutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δvsmmutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δvsmmutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor ofBacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs ofV. splendidus.
Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation
Authors: Kvist Malin , Hancock Viktoria , Klemm Per
Published: October 2008

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general.
Identification of a Novel Genomic Island Specific to Hospital-Acquired Clonal Complex 17
Enterococcus faecium
Isolates
Authors: Heikens Esther , van Schaik Willem , Leavis Helen L. , Bonten Marc J. M. , Willems Rob J. L.
Published: October 2008

Hospital-acquired clonal complex 17 (CC17)
Enterococcus faecium
strains are genetically distinct from indigenous strains and are enriched with resistance genes and virulence genes. We identified a genomic island in CC17
E. faecium
tentatively encoding a metabolic pathway involved in carbohydrate transport and metabolism, which may provide a competitive advantage over the indigenous
E. faecium
microbiota.
Fast Sterility Assessment by Germinable-Endospore Biodosimetry
Authors: Yung Pun To , Ponce Adrian
Published: October 2008

The increased demand for sterile products has created the need for rapid technologies capable of validating the hygiene of industrial production processes.
Bacillus
endospores are in standard use as biological indicators for evaluating the effectiveness of sterilization processes. Currently, culture-based methods, requiring more than 2 days before results become available, are employed to verify endospore inactivation. We describe a rapid, microscopy-based endospore viability assay (μEVA) capable of enumerating germinable endospores in less than 15 min. μEVA employs time-gated luminescence microscopy to enumerate single germinable endospores via terbium-dipicolinate (Tb-DPA) luminescence, which is triggered under UV excitation as 10
8
DPA molecules are released during germination on agarose containing Tb
3+
and a germinant (e.g.,
l
-alanine). Inactivation of endospore populations to sterility was monitored with μEVA as a function of thermal and UV dosage. A comparison of culturing results yielded nearly identical decimal reduction values, thus validating μEVA as a rapid biodosimetry method for monitoring sterilization processes. The simple Tb-DPA chemical test for germinability is envisioned to enable fully automated instrumentation for in-line monitoring of hygiene in industrial production processes.
Inferences about the Global Population Structures of
Cryptosporidium parvum
and
Cryptosporidium hominis
Authors: Tanrıverdi Sultan , Grinberg Alex , Chalmers Rachel M. , Hunter Paul R. , Petrovic Zorana , Akiyoshi Donna E. , London Eric , Zhang Linghui , Tzipori Saul , Tumwine James K. , Widmer Giovanni
Published: October 2008

Cryptosporidium parvum
and
Cryptosporidium hominis
are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of
C. parvum
and
C. hominis
isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.
Quantification and Genotyping of Torque Teno Virus at a Wastewater Treatment Plant in Japan
Authors: Haramoto Eiji , Katayama Hiroyuki , Ohgaki Shinichiro
Published: October 2008

Torque teno virus (TTV) DNA was quantitatively detected in influent and effluent samples collected from a wastewater treatment plant in Japan, with the highest concentration being 4.8 × 10
4
copies/liter. Genogroup-specific nested PCR demonstrated that TTV of genogroup 3 was the most abundant in wastewater among the five genogroups tested.
Regulation of Aromatic Alcohol Production in
Candida albicans
Authors: Ghosh Suman , Kebaara Bessie W. , Atkin Audrey L. , Nickerson Kenneth W.
Published: October 2008

Colonization by the fungal pathogen
Candida albicans
varies significantly, depending upon the pH and availability of oxygen. Because of our interest in extracellular molecules as potential quorum-sensing molecules, we examined the physiological conditions which regulate the production of the aromatic alcohols, i.e., phenethyl alcohol, tyrosol, and tryptophol. The production of these fusel oils has been well studied for
Saccharomyces cerevisiae
. Our data show that aromatic alcohol yields for
C. albicans
are determined by growth conditions. These conditions include the availability of aromatic amino acids, the pH, oxygen levels, and the presence of ammonium salts. For example, for wild-type
C. albicans
, tyrosol production varied 16-fold merely with the inclusion of tyrosine or ammonium salts in the growth medium. Aromatic alcohol production also depends on the transcription regulator Aro80p. Our results are consistent with aromatic alcohol production—aromatic transaminases (gene products for
ARO8
and
ARO9
), aromatic decarboxylase (
ARO10
), and alcohol dehydrogenase (
ADH
)—via the fusel oil pathway. The expression of
ARO8, ARO9
, and
ARO10
is also pH dependent.
ARO8
and
ARO9
were alkaline upregulated, while
ARO10
was alkaline downregulated. The alkaline-dependent change in expression of
ARO8
was Rim101 independent, while the expression of
ARO9
was Rim101 dependent.
Comparison of Electrode Reduction Activities of
Geobacter sulfurreducens
and an Enriched Consortium in an Air-Cathode Microbial Fuel Cell
Authors: Ishii Shun'ichi , Watanabe Kazuya , Yabuki Soichi , Logan Bruce E. , Sekiguchi Yuji
Published: October 2008

An electricity-generating bacterium,
Geobacter sulfurreducens
PCA, was inoculated into a single-chamber, air-cathode microbial fuel cell (MFC) in order to determine the maximum electron transfer rate from bacteria to the anode. To create anodic reaction-limiting conditions, where electron transfer from bacteria to the anode is the rate-limiting step, anodes with electrogenic biofilms were reduced in size and tests were conducted using anodes of six different sizes. The smallest anode (7 cm
2
, or 1.5 times larger than the cathode) achieved an anodic reaction-limiting condition as a result of a limited mass of bacteria on the electrode. Under these conditions, the limiting current density reached a maximum of 1,530 mA/m
2
, and power density reached a maximum of 461 mW/m
2
. Per-biomass efficiency of the electron transfer rate was constant at 32 fmol cell
−1
day
−1
(178 μmol g of protein
−1
min
−1
), a rate comparable to that with solid iron as the electron acceptor but lower than rates achieved with fumarate or soluble iron. In comparison, an enriched electricity-generating consortium reached 374 μmol g of protein
−1
min
−1
under the same conditions, suggesting that the consortium had a much greater capacity for electrode reduction. These results demonstrate that per-biomass electrode reduction rates (calculated by current density and biomass density on the anode) can be used to help make better comparisons of electrogenic activity in MFCs.
Comparative Characterization of the Microbial Diversities of an Artificial Microbialite Model and a Natural Stromatolite
Authors: Havemann Stephanie A. , Foster Jamie S.
Published: October 2008

Microbialites are organosedimentary structures that result from the trapping, binding, and lithification of sediments by microbial mat communities. In this study we developed a model artificial microbialite system derived from natural stromatolites, a type of microbialite, collected from Exuma Sound, Bahamas. We demonstrated that the morphology of the artificial microbialite was consistent with that of the natural system in that there was a multilayer community with a pronounced biofilm on the surface, a concentrated layer of filamentous cyanobacteria in the top 5 mm, and a lithified layer of fused oolitic sand grains in the subsurface. The fused grain layer was comprised predominantly of the calcium carbonate polymorph aragonite, which corresponded to the composition of the Bahamian stromatolites. The microbial diversity of the artificial microbialites and that of natural stromatolites were also compared using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. The ARISA profiling indicated that the Shannon indices of the two communities were comparable and that the overall diversity was not significantly lower in the artificial microbialite model. Bacterial clone libraries generated from each of the three artificial microbialite layers and natural stromatolites indicated that the cyanobacterial and crust layers most closely resembled the ecotypes detected in the natural stromatolites and were dominated by
Proteobacteria
and
Cyanobacteria
. We propose that such model artificial microbialites can serve as experimental analogues for natural stromatolites.
Proteome-Based Comparative Analyses of Growth Stages Reveal New Cell Cycle-Dependent Functions in the Predatory Bacterium
Bdellovibrio bacteriovorus
Authors: Dori-Bachash Mally , Dassa Bareket , Pietrokovski Shmuel , Jurkevitch Edouard
Published: October 2008

Bdellovibrio
and like organisms are obligate predators of bacteria that are ubiquitously found in the environment. Most exhibit a peculiar dimorphic life cycle during which free-swimming attack-phase (AP) cells search for and invade bacterial prey cells. The invader develops in the prey as a filamentous polynucleoid-containing cell that finally splits into progeny cells. Therapeutic and biocontrol applications of
Bdellovibrio
in human and animal health and plant health, respectively, have been proposed, but more knowledge of this peculiar cell cycle is needed to develop such applications. A proteomic approach was applied to study cell cycle-dependent expression of the
Bdellovibrio bacteriovorus
proteome in synchronous cultures of a facultative host-independent (HI) strain able to grow in the absence of prey. Results from two-dimensional gel electrophoresis, mass spectrometry, and temporal expression of selected genes in predicted operons were analyzed. In total, about 21% of the in silico predicted proteome was covered. One hundred ninety-six proteins were identified, including 63 hitherto unknown proteins and 140 life stage-dependent spots. Of those, 47 were differentially expressed, including chemotaxis, attachment, growth- and replication-related, cell wall, and regulatory proteins. Novel cell cycle-dependent adhesion, gliding, mechanosensing, signaling, and hydrolytic functions were assigned. The HI model was further studied by comparing HI and wild-type AP cells, revealing that proteins involved in DNA replication and signaling were deregulated in the former. A complementary analysis of the secreted proteome identified 59 polypeptides, including cell contact proteins and hydrolytic enzymes specific to predatory bacteria.
yKu70/yKu80 and Rif1 Regulate Silencing Differentially at Telomeres in
Candida glabrata
Authors: Rosas-Hernández Lluvia L. , Juárez-Reyes Alejandro , Arroyo-Helguera Omar E. , De Las Peñas Alejandro , Pan Shih-Jung , Cormack Brendan P. , Castaño Irene
Published: October 2008

Candida glabrata
, a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the
EPA
genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the
EPA
family, we have assessed the importance of
C. glabrata
orthologues of known regulators of subtelomeric silencing in
Saccharomyces cerevisiae.
To this end, we used a series of strains containing insertions of the reporter
URA3
gene within different intergenic regions throughout four telomeres of
C. glabrata
. Using these reporter strains, we have assessed the roles of
SIR2
,
SIR3
,
SIR4
,
HDF1
(yKu70),
HDF2
(yKu80), and
RIF1
in mediating silencing at four
C. glabrata
telomeres. We found that, whereas the
SIR
proteins are absolutely required for silencing of the reporter genes and the native subtelomeric
EPA
genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a
cis
element adjacent to the
EPA3
locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the
C. glabrata
telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements.
Genetic Analysis of the
Enterococcus
Vancomycin Resistance Conjugative Plasmid pHTβ: Identification of the Region Involved in Cell Aggregation and
traB
, a Key Regulator Gene for Plasmid Transfer and Cell Aggregation
Authors: Tomita Haruyoshi , Ike Yasuyoshi
Published: October 2008

The
Enterococcus
plasmid pHTβ (63.7 kbp) is a pheromone-independent, highly conjugative pMG1-like plasmid that carries a Tn
1546
-like transposon encoding vancomycin resistance. The transfer-related regions (Tra I, Tra II, and Tra III) containing
oriT
and a putative nickase gene (
traI
) have previously been identified in pHTβ, and in this study, we found that the plasmid conferred the ability to self-aggregate on the host strain
Enterococcus faecalis
FA2-2. A region where mutation resulted in the impairment of aggregation was identified and mapped to a point upstream of the transfer-related Tra I region. This region consisted of an approximately 6-kbp segment that contained the five open reading frames (ORFs) ORF9 to ORF13. These ORFs are considered to encode the aggregation function, although the precise mode of action of each ORF has not yet been elucidated. An in-frame deletion mutant of ORF10 resulted in reduced aggregation and decreased transfer frequency in broth mating. Transcription analysis of the aggregation region showed that the five ORFs from ORF9 to ORF13 form an operon structure, and a long transcript that started from a promoter region located upstream of ORF9 was identified. Tra II spans a 1.7-kbp region containing ORF56 and ORF57. Tn
917
-
lac
insertions into or an in-frame deletion mutant of ORF56 (187 amino acids) resulted in impaired transfer and aggregation. The cloned ORF56 complemented these functions in
trans
. The transcription levels of ORF10 and ORF13 were reduced in the in-frame mutants of ORF56, but this reduction was complemented by a cloned ORF56 in
trans
. The results indicated that ORF56 positively regulated the aggregation and plasmid transfer in the host strain, and ORF56 was designated
traB
.