bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Operating Parameters on the Current Density Distribution in Proton Exchange Membrane Fuel Cells
Authors: Zhang Y. , Mawardi A. , Pitchumani R.
Published: November 2006

During the operation of a proton exchange membrane (PEM) fuel cell, significant variation of the local current density could exist across the cell causing sharp temperature and stress gradients in certain points, and affecting the water management, all of which severely impact the cell performance and reliability. The variation of local current density is a critical issue in the performance of PEM fuel cell, and is influenced by the operating conditions. This article presents a model-assisted parametric design with the objective of determining the operating conditions which maximize the fuel cell performance while maintaining a level of uniformity in the current density distribution. A comprehensive two-dimensional model is adopted to simulate the species transport and electrochemical phenomena in a PEM fuel cell. Numerical simulations are performed for over a wide range of operating conditions to analyze the effects of various operating parameters on the variation of local current density of the fuel cell, and to develop design windows which serve as guideline in the design for maximum power density, minimum reactant stoichiometry, and uniform current density distribution.
Fuel Cells as Energy Sources for Future Mobile Devices
Authors: Tasa Sari , Aapro Teppo
Published: November 2006

Mobile device manufacturers would like to provide totally wireless solutions—including charging. Future multimedia devices need to have longer operation times as simultaneously they require more power. Device miniaturization leaves less volumetric space available also for the energy source. The energy density of the Li-ion batteries is high, and continuously developed, but not at the same speed as the demand from devices. Fuel cells can be one possible solution to power mobile devices without connection to the mains grid, but they will not fit to all use cases. The fuel cell system includes a core unit, fuel system, controls, and battery to level out peaks. The total energy efficiency is the sum of the performance of the whole system. The environmental performance of the fuel cell system cannot be determined yet. Regulatory and standardization work is on-going and driving the fuel cell technology development. The main target is in safety, which is very important aspect for energy technologies. The outcomes will also have an effect on efficiency, cost, design, and environmental performance. Proper water, thermal, airflow, and fuel management of the fuel cell system combined with mechanical durability and reliability are the crucial enablers for stable operation required from the integrated power source of a mobile device. Reliability must be on the same level as the reliability of the device the energy source is powering; this means years of continuous operation time. Typically, the end-users are not interested of the enabling technologies nor understand the usage limits. They are looking for easy to use devices to enhance their daily life. Fuel cell technology looks promising but there are many practical issues to be solved.
Biosyngas Utilization in Solid Oxide Fuel Cells With Ni∕GDC Anodes
Authors: Ouweltjes J. P. , Aravind P. V. , Woudstra N. , Rietveld G.
Published: November 2006

The combination of biomass gasification systems with fuel cells promises adequate systems for sustainable, decentralized energy conversion. Especially high temperature fuel cells are suited for this task because of their higher tolerance to impurities, their internal steam reforming potential, and favorable thermal integration possibilities. This paper presents the results of biosyngas utilization in solid oxide fuel cells with Ni∕GDC anodes at 850 and 920°C. The relation between the fuel composition and the electrochemical performance is discussed, as well as the impact of sulfur up to a concentration of 9ppmH2S. The investigations have made clear that Ni∕GDC anodes can be operated within a wide range of biosyngas compositions. Sulfur has appeared to deactivate the anode for methane reforming. The oxidation of hydrogen and carbon monoxide are insensitive to sulfur, suggesting that both nickel and GDC are active electrocatalysts.
Highly Efficient Conversion of Ammonia in Electricity by Solid Oxide Fuel Cells
Authors: Dekker N. J. J. , Rietveld G.
Published: November 2006

Hydrogen is the fuel for fuel cells with the highest cell voltage. A drawback for the use of hydrogen is the low energy density storage capacity, even at high pressures. Liquid fuels such as gasoline and methanol have a high energy density but lead to the emission of the greenhouse gas CO2. Ammonia could be the ideal bridge fuel, having a high energy density at relative low pressure and no (local) CO2 emission. Ammonia as a fuel for the solid oxide fuel cell (SOFC) appears to be very attractive, as shown by cell tests with electrolyte supported cells (ESC) as well as anode supported cells (ASC) with an active area of 81cm2. The cell voltage was measured as function of the electrical current, temperature, gas composition and ammonia (NH3) flow. With NH3 as fuel, electrical cell efficiencies up to 70% (LHV) can be achieved at 0.35A∕cm2 and 60% (LHV) at 0.6A∕cm2. The cell degradation during 3000 h of operation was comparable with H2 fueled measurements. Due to the high temperature and the catalytic active Ni∕YSZ anode, NH3 cracks at the anode into H2 and N2 with a conversion of >99.996%. The high NH3 conversion is partly due to the withdrawal of H2 by the electrochemical cell reaction. The remaining NH3 will be converted in the afterburner of the system. The NOx outlet concentration of the fuel cell is low, typically <0.5ppm at temperatures below 950°C and around 4ppm at 1000°C. A SOFC system fueled with ammonia is relative simple compared with a carbon containing fuel, since no humidification of the fuel is necessary. Moreover, the endothermic ammonia cracking reaction consumes part of the heat produced by the fuel cell, by which less cathode cooling air is required compared with H2 fueled systems. Therefore, the system for a NH3 fueled SOFC will have relatively low parasitic power losses and relative small heat exchangers for preheating the cathode air flow.
Role of Branched-Chain Fatty Acids in pH Stress Tolerance in
Listeria monocytogenes
Authors: Giotis Efstathios S. , McDowell David A. , Blair Ian S. , Wilkinson Brian J.
Published: November 2006

In alkaline conditions,
Listeria monocytogenes
cells develop higher proportions of branched-chain fatty acids (FAs), including more anteiso forms. In acid conditions, the opposite occurs. Reduced growth of pH-sensitive mutants at adverse pH (5.0/9.0) was alleviated by the addition of 2-methylbutyrate (an anteiso-FA precursor), suggesting that anteiso-FAs are important in adaptation to adverse pH. The balance between anteiso- and iso-FAs may be more important than changes in the amounts and/or degrees of saturation of FAs in pH adaptation.
Role of Zearalenone Lactonase in Protection of
Gliocladium roseum
from Fungitoxic Effects of the Mycotoxin Zearalenone
Authors: Utermark Jan , Karlovsky Petr
Published: November 2006

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of
Fusarium
. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of ≤10 μg/ml. The fungitoxic effect declined in the order zearalenone > α-zearalenol > β-zearalenol. The mycoparasitic fungus
Gliocladium roseum
produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of
G. roseum
was not inhibited by zearalenone, and the lactonase may protect
G. roseum
from the toxic effects of this mycotoxin. We inactivated
zes2
, the gene encoding zearalenone lactonase in
G. roseum
, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of
Agrobacterium tumefaciens
-mediated genetic transformation. The
zes2
disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.
Functional Analysis of
Burkholderia cepacia
Genes
bceD
and
bceF
, Encoding a Phosphotyrosine Phosphatase and a Tyrosine Autokinase, Respectively: Role in Exopolysaccharide Biosynthesis and Biofilm Formation
Authors: Ferreira Ana S. , Leitão Jorge H. , Sousa Sílvia A. , Cosme Ana M. , Sá-Correia Isabel , Moreira Leonilde M.
Published: November 2006

The biosynthesis of the exopolysaccharide (EPS) cepacian by
Burkholderia cepacia
complex strains requires the 16.2-kb
bce
cluster of genes. Two of the clustered genes,
bceD
and
bceF
, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate
p
-nitrophenyl phosphate (PNPP), BceD exhibited a
V
max
of 8.8 μmol of PNPP min
−1
mg
−1
and a

K
m

of 3.7 mM PNPP at 30°C. The disruption of
bceF
resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the
bceD
mutant. The exopolysaccharide produced by the
bceD
mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by
bceD
and
bceF
mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.
Molecular Analysis of Arsenate-Reducing Bacteria within Cambodian Sediments following Amendment with Acetate
Authors: Lear G. , Song B. , Gault A. G. , Polya D. A. , Lloyd J. R.
Published: November 2006

The health of millions is threatened by the use of groundwater contaminated with sediment-derived arsenic for drinking water and irrigation purposes in Southeast Asia. The microbial reduction of sorbed As(V) to the potentially more mobile As(III) has been implicated in release of arsenic into groundwater, but to date there have been few studies of the microorganisms that can mediate this transformation in aquifers. With the use of stable isotope probing of nucleic acids, we present evidence that the introduction of a proxy for organic matter (
13
C-labeled acetate) stimulated As(V) reduction in sediments collected from a Cambodian aquifer that hosts arsenic-rich groundwater. This was accompanied by an increase in the proportion of prokaryotes closely related to the dissimilatory As(V)-reducing bacteria
Sulfurospirillum
strain NP-4 and
Desulfotomaculum auripigmentum
. As(V) respiratory reductase genes (
arrA
) closely associated with those found in
Sulfurospirillum barnesii
and
Geobacter uraniumreducens
were also detected in active bacterial communities utilizing
13
C-labeled acetate in microcosms. This study suggests a direct link between inputs of organic matter and the increased prevalence and activity of organisms which transform As(V) to the potentially more mobile and thus hazardous As(III) via dissimilatory As(V) reduction.
Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays
Authors: Stedtfeld Robert D. , Wick Lukas M. , Baushke Samuel W. , Tourlousse Dieter M. , Herzog Amanda B. , Xia Yongmei , Rouillard Jean Marie , Klappenbach Joel A. , Cole James R. , Gulari Erdogan , Tiedje James M. , Hashsham Syed A.
Published: November 2006

Dangling
ends and surface-proximal tails of gene targets influence probe-target
duplex formation and affect the signal intensity of probes on
diagnostic microarrays. This phenomenon was evaluated using an
oligonucleotide microarray containing 18-mer probes corresponding to
the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic
and PCR-amplified gene targets. Signal intensities for Klenow/random
primer-labeled 16S rRNA gene targets were dissimilar from those for
45-mer synthetic targets for nearly 73% of the probes tested.
Klenow/random primer-labeled targets resulted in an interaction with a
complex mixture of 16S rRNA genes (used as the background) 3.7 times
higher than the interaction of 45-mer targets with the same mixture. A
7-base-long dangling end sequence with perfect homology to another
single-stranded background DNA sequence was sufficient to produce a
cross-hybridization signal that was as strong as the signal obtained by
the probe-target duplex itself. Gibbs free energy between the target
and a well-defined background was found to be a better indicator of
hybridization signal intensity than the sequence or length of the
dangling end alone. The dangling end (Gibbs free energy of −7.6
kcal/mol) was found to be significantly more prone to target-background
interaction than the surface-proximal tail (Gibbs free energy of−
64.5 kcal/mol). This study underlines the need for careful
target preparation and evaluation of signal intensities for diagnostic
arrays using 16S rRNA and other gene targets due to the potential for
target interaction with a complex
background.
Phylogenetic Analysis of the pPT23A Plasmid Family of
Pseudomonas syringae
Authors: Ma Zhonghua , Smith James J. , Zhao Youfu , Jackson Robert W. , Arnold Dawn L. , Murillo Jesús , Sundin George W.
Published: November 2006

The pPT23A plasmid family of
Pseudomonas syringae
contains members that contribute to the ecological and pathogenic fitness of their
P. syringae
hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from
P. syringae
. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (
repA
), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the
P. syringae
hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (
gyrB
) and primary sigma factor (
rpoD
), however, were strongly associated with genomospecies of
P. syringae
. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among
P. syringae
pathovars.
Sulfur-Oxidizing Bacteria in Soap Lake (Washington State), a Meromictic, Haloalkaline Lake with an Unprecedented High Sulfide Content
Authors: Sorokin Dimitry Y. , Foti Mirjam , Pinkart Holly C. , Muyzer Gerard
Published: November 2006

Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genusThioalkalimicrobium, which are common inhabitants of soda lakes. A dense population ofThioalkalimicrobium(up to 107cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. TwelveThioalkalimicrobiumstrains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related toT. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from knownThioalkalimicrobiumspecies and unique to Soap Lake. It represented a new species, for which we suggest the nameThioalkalimicrobium microaerophilumsp. nov.
Use of Quantum Dot Luminescent Probes To Achieve Single-Cell Resolution of Human Oral Bacteria in Biofilms
Authors: Chalmers Natalia I. , Palmer Robert J. , Du-Thumm Laurence , Sullivan Richard , Shi Wenyuan , Kolenbrander Paul E.
Published: November 2006

Oral biofilms are multispecies communities, and in their nascent stages of development, numerous bacterial species engage in interspecies interactions. Better insight into the spatial relationship between different species and how species diversity increases over time can guide our understanding of the role of interspecies interactions in the development of the biofilms. Quantum dots (QD) are semiconductor nanocrystals and have emerged as a promising tool for labeling and detection of bacteria. We sought to apply QD-based primary immunofluorescence for labeling of bacterial cells with in vitro and in vivo biofilms and to compare this approach with the fluorophore-based primary immunofluorescence approach we have used previously. To investigate QD-based primary immunofluorescence as the means to detect distinct targets with single-cell resolution, we conjugated polyclonal and monoclonal antibodies to the QD surface. We also conducted simultaneous QD conjugate-based and fluorophore conjugate-based immunofluorescence and showed that these conjugates were complementary tools in immunofluorescence applications. Planktonic and biofilm cells were labeled effectively by considering two factors: the final nanomolar concentration of QD conjugate and the amount of antibody conjugated to the QD, which we define as the degree of labeling. These advances in the application of QD-based immunofluorescence for the study of biofilms in vitro and in vivo will help to define bacterial community architecture and to facilitate investigations of interactions between bacterial species in these communities.
Ste12 Transcription Factor Homologue CpST12 Is Down-Regulated by HypovirusInfection and Required for Virulence and Female Fertility of the Chestnut Blight Fungus
Cryphonectria parasitica
Authors: Deng Fuyou , Allen Todd D. , Nuss Donald L.
Published: November 2006

A
putative homologue of the
Saccharomyces cerevisiae
Ste12
transcription factor was identified in a series of expressed sequence
tag-based microarray analyses as being down-regulated in strains of the
chestnut blight fungus,
Cryphonectria parasitica
, infected by
virulence-attenuating hypoviruses. Cloning of the corresponding gene,
cpst12
, confirmed a high level of similarity to Ste12
homologues of other filamentous fungi. Disruption of
cpst12
resulted in no alterations in in vitro growth characteristics or colony
morphology and an increase in the production of asexual spores,
indicating that CpST12 is dispensable for vegetative growth and
conidiation on artificial medium. However, the disruption mutants
showed a very substantial reduction in virulence on chestnut tissue and
a complete loss of female fertility, two symptoms normally conferred by
hypovirus infection. Both virulence and female fertility were restored
by complementation with the wild-type
cpst12
gene. Analysis of
transcriptional changes caused by
cpst12
gene disruption with
a custom
C. parastica
cDNA microaray chip identified 152
responsive genes. A significant number of these putative
CpST12-regulated genes were also responsive to hypovirus infection.
Thus,
cpst12
encodes a cellular transcription factor, CpST12,
that is down-regulated by hypovirus infection and required for female
fertility, virulence and regulated expression of a subset of hypovirus
responsive host
genes.
Radial Mass Density, Charge, and Epitope Distribution in theCryptococcus neoformansCapsule
Authors: Maxson Michelle E. , Dadachova Ekaterina , Casadevall Arturo , Zaragoza Oscar
Published: November 2006

Exposure ofCryptococcus neoformanscells to gamma radiation results in a gradual release of capsular polysaccharide, in a dose-dependent manner. This method allows the systematic exploration of different capsular regions. Using this methodology, capsule density was determined to change according to the radial distribution of glucuronoxylomannan and total polysaccharide, becoming denser at the inner regions of the capsule. Scanning electron microscopy of cells following gamma radiation treatment confirmed this finding. The zeta potential of the capsule also increased as the capsule size decreased. However, neither charge nor density differences were correlated with any change in sugar composition (xylose, mannose, and glucuronic acid) in the different capsular regions, since the proportions of these sugars remained constant throughout the capsule. Analysis of the capsular antigenic properties by monoclonal antibody binding and Scatchard analysis revealed fluctuations in the binding affinity within the capsule but not in the number of antibody binding sites, suggesting that the spatial organization of high- and low-affinity epitopes within the capsule changed according to radial position. Finally, evidence is presented that the structure of the capsule changes with capsule age, since the capsule of older cells became more resistant to gamma radiation-induced ablation. In summary, the capsule ofC. neoformansis heterogeneous in its spatial distribution and changes with age. Furthermore, our results suggest several mechanisms by which the capsule may protect the fungal cell against exogenous environmental factors.
Vesicular Polysaccharide Export in
Cryptococcus neoformans
Is a Eukaryotic Solution to the Problem of Fungal Trans-Cell Wall Transport
Authors: Rodrigues Marcio L. , Nimrichter Leonardo , Oliveira Débora L. , Frases Susana , Miranda Kildare , Zaragoza Oscar , Alvarez Mauricio , Nakouzi Antonio , Feldmesser Marta , Casadevall Arturo
Published: November 2006

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of
Cryptococcus neoformans
, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that
C. neoformans
produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of
C. neoformans
whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.
Pseudomonas aeruginosa
LecB Is Involved in Pilus Biogenesis and Protease IV Activity but Not in Adhesion to Respiratory Mucins
Authors: Sonawane Avinash , Jyot Jeevan , Ramphal Reuben
Published: November 2006

Pseudomonas aeruginosa
expresses two lectins which are implicated in adhesion and biofilm formation. In this study, we demonstrate that
P. aeruginosa
LecB is involved in pilus biogenesis and proteolytic activity. Moreover, neither lectin was involved in adhesion to human tracheobronchial mucin. We infer that some of the ascribed functions are secondary effects on other systems rather than effects of the lectins themselves.
Pathway Confirmation and Flux Analysis of Central Metabolic Pathways in
Desulfovibrio vulgaris
Hildenborough using Gas Chromatography-Mass Spectrometry and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
Authors: Tang Yinjie , Pingitore Francesco , Mukhopadhyay Aindrila , Phan Richard , Hazen Terry C. , Keasling Jay D.
Published: November 2006

Flux distribution in central metabolic pathways of
Desulfovibrio vulgaris
Hildenborough was examined using
13
C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (
S
)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in
D. vulgaris
and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.
Role of a Putative Polysaccharide Locus in
Bordetella
Biofilm Development
Authors: Parise Gina , Mishra Meenu , Itoh Yoshikane , Romeo Tony , Deora Rajendar
Published: November 2006

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by
Bordetella
spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-β-1,6-
N
-acetyl-
d
-glucosamine (poly-β-1,6-GlcNAc), we provide evidence for the production of poly-β-1,6-GlcNAc by various
Bordetella
species (
Bordetella bronchiseptica
,
B. pertussis
, and
B. parapertussis
) and its role in their biofilm development. We have investigated the role of a
Bordetella
locus, here designated
bpsABCD
, in biofilm formation. The
bps
(
Bordetella
polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-β-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the
bps
locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of
Bordetella
biofilms.
AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in
Mycobacterium tuberculosis
Authors: Daniel Jaiyanth , Oh Tae-Jin , Lee Chang-Muk , Kolattukudy Pappachan E.
Published: November 2006

The
Mycobacterium tuberculosis
acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The
M. tuberculosis
genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with
accD6
located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of
accA3
,
accD4
,
accD5
, and
accD6
are expressed at high levels during the exponential growth phases of
M. tuberculosis
in vitro. Microarray analysis of
M. tuberculosis
transcripts indicated that the transcripts for
accA3
,
accD4
,
accD5
,
accD6
, and
accE
were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (α
3
) and AccD6 (β
6
) in
E. coli
and purified them by affinity chromatography. We report here that reconstitution of the α
3
-β
6
complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the α
3
-β
6
complex was inhibited by the ε subunit. The α
3
-β
6
carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the β
6
subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.
The
Streptococcus mutans vicX
Gene Product Modulates
gtfB
/
C
Expression, Biofilm Formation, Genetic Competence, and Oxidative Stress Tolerance
Authors: Senadheera M. Dilani , Lee Andrew W. C. , Hung David C. I. , Spatafora Grace A. , Goodman Steven D. , Cvitkovitch Dennis G.
Published: November 2006

Streptococcus mutans
is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of
S. mutans
. In this study, we focused on the
vicX
gene of the
vicRKX
tricistronic operon. To characterize
vicX
, we constructed a nonpolar deletion mutation in the
vicX
coding region in
S. mutans
UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of
vicX
(
P
< 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in
S. mutans
. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly,
vicX
-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (
gtfB
/
C
) expression compared with the expression in the wild type (
P
< 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an
S. mutans
GS5-derived
vicX
null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking
gtfB
and/or
gtfC
. Also, disruption of
vicX
reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (
P
< 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the
vicX
gene product that can impact
S. mutans
pathogenicity.
Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains
Authors: Hill K. K. , Smith T. J. , Helma C. H. , Ticknor L. O. , Foley B. T. , Svensson R. T. , Brown J. L. , Johnson E. A. , Smith L. A. , Okinaka R. T. , Jackson P. J. , Marks J. D.
Published: November 2006

Clostridium botulinum
is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174
C. botulinum
strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.
Biological Relevance of Colony Morphology and Phenotypic Switching by
Burkholderia pseudomallei
Authors: Chantratita Narisara , Wuthiekanun Vanaporn , Boonbumrung Khaemaporn , Tiyawisutsri Rachaneeporn , Vesaratchavest Mongkol , Limmathurotsakul Direk , Chierakul Wirongrong , Wongratanacheewin Surasakdi , Pukritiyakamee Sasithorn , White Nicholas J. , Day Nicholas P. J. , Peacock Sharon J.
Published: November 2006

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism,
Burkholderia pseudomallei
, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical
B. pseudomallei
isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed
B. pseudomallei
morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.
Characterization of OpdH, a
Pseudomonas aeruginosa
Porin Involved in the Uptake of Tricarboxylates
Authors: Tamber Sandeep , Maier Elke , Benz Roland , Hancock Robert E. W.
Published: November 2006

The
Pseudomonas aeruginosa
outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of
cis
-aconitate. Here we demonstrate that this porin may also enable
P. aeruginosa
to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the
opdH
gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.
Essential Bacterial Functions Encoded by Gene Pairs
Authors: Thomaides Helena B. , Davison Ella J. , Burston Lisa , Johnson Hazel , Brown David R. , Hunt Alison C. , Errington Jeffery , Czaplewski Lloyd
Published: November 2006

To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs inBacillus subtilisand attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.
Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic ArchaeonPyrococcus furiosus
Authors: Madding Lara S. , Michel Joshua K. , Shockley Keith R. , Conners Shannon B. , Epting Kevin L. , Johnson Matthew R. , Kelly Robert M.
Published: November 2006

The hyperthermophilic archaeonPyrococcus furiosusgenome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of theP. furiosusdynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in theP. furiosus20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.
Characterization ofFrancisella tularensisOuter Membrane Proteins
Authors: Huntley Jason F. , Conley Patrick G. , Hagman Kayla E. , Norgard Michael V.
Published: November 2006

Francisella tularensisis a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) ofF. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putativeF. tularensisouter membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins.F. tularensisOMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method forF. tularensisOM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization ofF. tularensisOMPs.
Physical and Functional Interactions between
Escherichia coli
MutY Glycosylase and Mismatch Repair Protein MutS
Authors: Bai Haibo , Lu A-Lien
Published: November 2006

Escherichia coli
MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSα (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in
E. coli
. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in
mutS
mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a
mutS mutY
mutant compared to a
mutS
mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.
Catalase (KatA) and Alkyl Hydroperoxide Reductase (AhpC) Have Compensatory Roles in Peroxide Stress Resistance and Are Required for Survival, Persistence, and Nasal Colonization inStaphylococcus aureus
Authors: Cosgrove Kate , Coutts Graham , Jonsson Ing-Marie , Tarkowski Andrej , Kokai-Kun John F. , Mond James J. , Foster Simon J.
Published: November 2006

Oxidative-stress resistance inStaphylococcus aureusis linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members,ahpC(alkylhydroperoxide reductase) andkatA(catalase), show compensatory regulation, with independent and linked functions. AnahpCmutation leads to increased H2O2resistance due to greaterkatAexpression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in akatAmutant. Mutation of bothkatAandahpCleads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, thekatA ahpCmutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability ofS. aureusto persist in the hospital environment and so contribute to the spread of human disease.
FATIGUE PROPERTIES OF HYBRID SURFACE MODIFIED SCM435H STEEL
Authors: KIKUCHI SHOICHI , FUKAZAWA KENGO , KOMOTORI JUN , SHIMIZU MASAO
Published: November 2006

In order to clarify the effects of nitriding and hybrid surface modification process combining nitriding and induction hardening on fatigue properties of SCM435H steel, high cycle fatigue tests were carried out with a rotational bending machine at room temperature. Observations of fracture surfaces and measurement of hardness and residual stress distributions were carried out to investigate the fracture mechanism. It was revealed that hybrid surface modification process generated a compressive residual stress field and hardened even at center of the specimen. Fatigue strength of hybrid surface modified specimens was much higher than that of substrate and nitrided specimens. This was because a transition of fracture mode from internal to surface fracture; fatigue fracture of nitrided specimens occurred at inside of the hardened layer, in the case of hybrid surface modified specimens, however, fatigue crack initiated at the surface of the specimen with higher hardness and higher compressive residual stress.
LOCAL VIBRATIONAL MODES AND THE OPTICAL ABSORPTION TAIL OF AMORPHOUS SILICON
Authors: LIM P. K. , TAM W. K.
Published: November 2006

Local vibrational modes of Si - H is an important research area in recent years. Local vibrational modes of chemical bonds between Si atom and other impurity atoms such as H and O in amorphous silicon films produced by radio frequency sputtering were studied by means of Fourier transform infrared spectroscopy. The concentrations of Si - H , Si - O and Si - C in the sample were calculated. It was found that the concentration of Si - H bond varied significantly when the material was annealed at temperatures Ta >600 K and tended to zero for Ta >1000 K . The optical absorption edge was also found to depend strongly on the thermal history of the amorphous semiconductor. A strong correlation between the optical absorption coefficient and the concentration of Si - H was also observed.