bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust Pose Estimation With an Outlier Diagnosis Based on a Relaxation of Rigid Body Constraints
Authors: Lin Yu , Tu Xiao-wei , Xi Fengfeng , Chan Vincent
Published: November 2012

In this paper, we propose a novel outlier diagnosis method for robust pose estimation of rigid body motions from outlier contaminated 3D point measurements. Due to incorrect correspondences in a cluttered measuring environment, observed point data are contaminated by outliers, which are unusual gross errors that lie out of an overall error distribution. Standard least-squares methods for pose estimation are highly sensitive to outliers. For this reason, an outlier diagnosis method is developed to preprocess measured point data prior to pose estimation. This diagnosis method detects and removes outliers based on a relaxation method with rigid body constraints of a rigid body. Simulations and experiments prove the effectiveness and advantages of high breakdown point and ease of implementation.
Piezoelectric Injectors for Automotive Applications: Modeling and Experimental Validation of Hysteretic Behavior and Temperature Effects
Authors: Altieri Leonardo , Tonoli Andrea
Published: November 2012

Direct acting piezoelectric injectors seem to be a promising alternative to the electromagnetic ones because they permit a continuous control of the aperture. This characteristic can improve the performances and minimize the emissions of diesel engines. To exploit the potentialities of this kind of actuation, it is necessary to minimize the effects of the hysteretic behavior of piezoelectric materials. For this reason, the behavior of the actuator has to be modeled taking this effect into account. Additionally, the effects of the temperature must be considered, given the particularly critical position of the injectors near the engine. A modeling approach of piezoelectric injectors, including hysteresis and temperature effects as well as the electromechanical dynamic, is described in this paper. The model is based on a linear finite element (FE) discretization of the piezoelectric stack and the injector case. The hysteretic behavior is included in a second step by means of additional nonlinear state equations while the temperature effects are taken into account considering the temperature dependence of the material characteristics. A dedicated test bench has then been realized and experimental tests have been performed on piezoelectric injectors, with driving voltages and temperatures commonly used in automotive environment. The collected data allow to tune the model and to verify its validity even out of the tuning conditions.
A New Robust Filtering Strategy for Linear Systems
Authors: Gadsden S. A. , Habibi S. R.
Published: November 2012

For linear and well-defined estimation problems with Gaussian white noise, the Kalman filter (KF) yields the best result in terms of estimation accuracy. However, the KF performance degrades and can fail in cases involving large uncertainties such as modeling errors in the estimation process. The smooth variable structure filter (SVSF) is a relatively new estimation strategy based on sliding mode theory and has been shown to be robust to modeling uncertainties. The SVSF makes use of an existence subspace and of a smoothing boundary layer to keep the estimates bounded within a region of the true state trajectory. Currently, the width of the smoothing boundary layer is chosen based on designer knowledge of the upper bound of modeling uncertainties, such as maximum noise levels and parametric errors. This is a conservative choice, as a more well-defined smoothing boundary layer will yield more accurate results. In this paper, the state error covariance matrix of the SVSF is used for the derivation of an optimal time-varying smoothing boundary layer. The robustness and accuracy of the new form of the SVSF was validated and compared with the KF and the standard SVSF by testing it on a linear electrohydrostatic actuator (EHA).
Recursive Identification of Nonlinear Cascade Systems With Time-Varying General Input Backlash
Authors: Vörös Jozef
Published: November 2012

Recursive identification of nonlinear cascade systems with a time-varying general input backlash and a linear dynamic system is presented. A new analytic form of general backlash characteristic description is used, where instead of the straight lines determining the upward and downward parts of backlash characteristic, general curves are considered. All the parameters in the cascade model equation are separated and their estimation is solved as a quasi-linear problem using the recursive least squares method with internal variable estimation. Simulation studies are included for more cascade systems with time-varying general input backlash.
Effect of Exhaust Gas Recirculation on Biodiesel Blend Level Estimation in Diesel Engines
Authors: Zhao Junfeng , Wang Junmin
Published: November 2012

Biodiesel is an alternative fuel derived from vegetable oils, animal fats, or other sources, and it can be made into biodiesel blends by mixing with conventional diesel. To achieve optimal engine combustion as well as minimal emissions with biodiesel blends, on-board blend level estimation system is one of the prerequisites. The paper investigates a blend level estimation system by evaluating exhaust oxygen fraction differences between diesel and biodiesel. In the system, a wideband oxygen sensor is utilized to measure the oxygen concentration. Then, research on the oxygen-content based biodiesel blend level estimation system is extended by taking the exhaust gas recirculation (EGR) into account. Since estimation accuracy under the lean conditions for this kind of system has been especially problematic, the effect of EGR on system estimation uncertainty is discussed. The theoretical formulation shows that oxygen consumption factor is an intrinsic indicator of fuels, which is not affected by the EGR level. However, the “shifting effect” caused by introducing EGR can help moving the estimation points into a range where not only the oxygen sensor but also the whole estimation system exhibits lower measurement uncertainty. Validations are provided by both simulation results based on a high-fidelity GT-Power engine model and experimental results on a medium-duty turbo-charged diesel engine platform.
Dynamic Optimization of Drivetrain Gear Ratio to Maximize Wind Turbine Power Generation—Part 2: Control Design
Authors: Hall John F. , Chen Dongmei
Published: November 2012

The cost of electrical power produced by small wind turbines impedes the use of this technology, which can otherwise provide power to millions of homes in rural regions worldwide. To encourage their use, small wind turbines must capture wind energy more effectively while avoiding increased equipment costs. A variable ratio gearbox (VRG) can provide this capability to the simple fixed-speed wind turbine through discrete operating speeds. This is the second of a two-part publication that focuses on the control of a VRG-enabled wind turbine. The first part presented a 100 kW fixed speed, wind turbine model, and a method for manipulating the VRG and mechanical brake to achieve full load operation. In this study, an optimal control algorithm is developed to maximize the power production in light of the limited brake pad life. Recorded wind data are used to develop a customized control design that is specific to a given site. Three decision-making modules interact with the wind turbine model developed in Part 1 to create possible VRG gear ratio (GR) combinations. Dynamic programming is applied to select the optimal combination and establish the operating protocol. The technique is performed on 20 different wind profiles. The results suggest an increase in wind energy production of nearly 10%.
Velocity Estimation of Motion Systems Based on Low-Resolution Encoders
Authors: Zhu Hongzhong , Sugie Toshiharu
Published: November 2012

This paper proposes a new approach to estimate the velocity of mechanical system in the case where the optical incremental encoder is used as the position sensor. First, the actual angular position is reconstructed via moving horizon polynomial fitting method by taking account of quantization feature and the plant dynamics. Then, the reconstruction signal is applied to a classical observer to obtain the velocity estimation. Its robustness against the position sensor resolution and the degree of the polynomial is discussed by some numerical examples. Experiments with very low-resolution encoder in low speed range also confirm its effectiveness.
Development of a Special Inertial Measurement Unit for UAV Applications
Authors: Hatamleh Khaled S. , Ma Ou , Flores-Abad Angel , Xie Pu
Published: November 2012

Dynamics modeling is becoming more and more important in the development and control of unmanned aerial vehicles (UAV). An accurate model of a vehicle requires good knowledge of the dynamics properties and motion states, which are usually estimated with the help of integrated inertial measurement units (IMUs). This work develops a special six degrees of freedom IMU, which has the capability of measuring the angular accelerations. This paper introduces the design of the new IMU along with its sensor models and calibration procedures. The work introduces two experimental methods to verify the calibrated IMU readings. The IMU was designed to support an on-line methodology to estimate the parameters of UAV’s dynamics model that is currently being developed by the authors.
Film Cooling Extraction Effects on the Aero-Thermal Characteristics of Rib Roughened Cooling Channel Flow
Authors: Cukurel Beni , Selcan Claudio , Arts Tony
Published: November 2012

The present study is geared towards quantifying the effects of film cooling holes on turbine internal cooling passages. In this regard, tests are conducted in a generic stationary model, with evenly distributed rib-type perturbators at 90 deg, constituting a passage blockage ratio of H/Dh = 0.3 and pitch-to-height ratio of P/H = 10. The 1/3H diameter surface-perpendicular film cooling holes are employed at a distance of 5/3H downstream of the preceding rib. Through liquid crystal thermometry measurements, the aero-thermal effects of a change in suction ratio are contrasted for various configurations (Re = 40,000 SR = 0–6), and compared with the analogous aerodynamic literature, enabling heat transfer distributions to be associated with distinct flow structures. At increased suction ratio, the size of the separation bubble downstream of the rib is observed to diminish, triggering globally an earlier reattachment; in addition to low-momentum hot fluid extraction via film cooling suction. Hence, in the presence of active flow extraction, higher overall heat transfer characteristics are observed throughout the channel. Moreover, the findings are generalized via friction factor and Nusselt number correlations, along with an analytical 20-pitch passage model. SR ∼ 3.5 is observed to provide favorable characteristics of pitch-to-pitch uniform suction ratio, lack of hot fluid ingestion and to sustain the highest passage averaged heat transfer.
Constrained Adjoint-Based Aerodynamic Shape Optimization of a Single-Stage Transonic Compressor
Authors: Walther Benjamin , Nadarajah Siva
Published: November 2012

This paper develops a discrete adjoint formulation for the constrained aerodynamic shape optimization in a multistage turbomachinery environment. The adjoint approach for viscous internal flow problems and the corresponding adjoint boundary conditions are discussed. To allow for a concurrent rotor/stator optimization, a nonreflective adjoint mixing-plane formulation is proposed. A sequential-quadratic programming algorithm is utilized to determine an improved airfoil shape based on the objective function gradient provided by the adjoint solution. The functionality of the proposed optimization method is demonstrated by the redesign of a midspan section of a single-stage transonic compressor. The objective is to maximize the isentropic efficiency while constraining the mass flow rate and the total pressure ratio.
Vaned Diffuser Induced Impeller Blade Vibrations in a High-Speed Centrifugal Compressor
Authors: Zemp Armin , Abhari Reza S.
Published: November 2012

Blade failure in turbomachinery is frequently caused by an excessive resonant response. Forced response of the blades typically originates from unsteady fluid structure interactions. This paper presents the experimental and computational results of a research effort focusing on the blade forced response in a high-speed centrifugal compressor caused by the downstream vaned diffuser. The potential field from the downstream vaned diffuser acts as an unsteady impeller relative circumferentially nonuniform disturbance. In this work the effect of varying the radial gap between impeller exit and diffuser vane leading edges was examined. Dynamic strain gauges, which were installed on the blade surfaces, were used to measure the forced response levels of the blades and to estimate the damping properties for different compressor operating conditions and vaneless gap dimensions. Unsteady fluid flow simulations were used to quantify the forcing function acting on the compressor blades due to impeller-diffuser interaction. The time-resolved blade pressure distribution showed the temporal evolution of the dynamic load on the blade surface caused by the diffuser's potential field. The magnitude of the vibratory stress levels was found to depend on the radial gap size, the blade damping properties, and on the compressor operating point. The variation of the radial gap size resulted in a shift of the impeller-diffuser interaction zone towards the main blade leading edge by up to 5% of the streamwise location.
Convolution-Based Trajectory Generation Methods Using Physical System Limits
Authors: Lee Geon , Kim Jinhyun , Choi Youngjin
Published: November 2012

This paper proposes two novel convolution-based trajectory generation methods using physical system limits such as maximum velocity, maximum acceleration, and maximum jerk. Convolution is a mathematical operation on two functions of an input function and a convoluted function, producing an output function that is typically viewed as a modified version of input function. Time duration parameters of the convoluted functions with a unit area are determined from the given physical system limits. The convolution-based trajectory generation methods to be proposed in this paper have three advantages; first, a continuously differentiable trajectory is simply obtained by applying successive convolution operations; second, a resultant trajectory is always generated satisfying the given physical system limits; third, the suggested methods have low computational burden thanks to recursive form of convolution operation. The suggested methods consider both zero and nonzero initial/terminal conditions. Finally, the effectiveness of the suggested methods is shown through numerical simulations.
Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes
Authors: Geszvain Kati , McCarthy James K. , Tebo Bradley M.
Published: November 2012

Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in
Pseudomonas putida
GB-1 has long remained unknown. To identify the
P. putida
GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (
mnxG
,
mofA
,
moxA
, and
mopA
). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11
Pseudomonas
species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.
Bifidobacterial Succession and Correlation Networks in a Large Unselected Cohort of Mothers and Their Children
Authors: Avershina E. , Storrø O. , Øien T. , Johnsen R. , Wilson R. , Egeland T. , Rudi K.
Published: November 2012

Bifidobacteria are a major microbial component of infant gut microbiota, which is believed to promote health benefits for the host and stimulate maturation of the immune system. Despite their perceived importance, very little is known about the natural development of and possible correlations between bifidobacteria in human populations. To address this knowledge gap, we analyzed stool samples from a randomly selected healthy cohort of 87 infants and their mothers with >90% of vaginal delivery and nearly 100% breast-feeding at 4 months. Fecal material was sampled during pregnancy, at 3 and 10 days, at 4 months, and at 1 and 2 years after birth. Stool samples were predicted to be rich in the species
Bifidobacterium adolescentis
,
B. bifidum
,
B. dentium
,
B. breve
, and
B. longum
. Due to high variation, we did not identify a clear age-related structure at the individual level. Within the population as a whole, however, there were clear age-related successions. Negative correlations between the
B. longum
group and
B. adolescentis
were detected in adults and in 1- and 2-year-old children, whereas negative correlations between
B. longum
and
B. breve
were characteristic for newborns and 4-month-old infants. The highly structured age-related development of and correlation networks between bifidobacterial species during the first 2 years of life mirrors their different or competing nutritional requirements, which in turn may be associated with specific biological functions in the development of healthy gut.
Gene Flow, Recombination, and Selection in Cyanobacteria: Population Structure of Geographically Related Planktothrix Freshwater Strains
Authors: Sogge Hanne , Rohrlack Thomas , Rounge Trine B. , Sønstebø Jørn Henrik , Tooming-Klunderud Ave , Kristensen Tom , Jakobsen Kjetill S.
Published: November 2012

Several
Planktothrix
strains, each producing a distinct oligopeptide profile, have been shown to coexist within Lake Steinsfjorden (Norway). Using nonribosomal peptide synthetase (NRPS) genes as markers, it has been shown that the
Planktothrix
community comprises distinct genetic variants displaying differences in bloom dynamics, suggesting a
Planktothrix
subpopulation structure. Here, we investigate the
Planktothrix
variants inhabiting four lakes in southeast of Norway utilizing both NRPS and non-NRPS genes. Phylogenetic analyses showed similar topologies for both NRPS and non-NRPS genes, and the lakes appear to have similar structuring of
Planktothrix
genetic variants. The structure of distinct variants was also supported by very low genetic diversity within variants compared to the between-variant diversity. Incongruent topologies and split decomposition revealed recombination events between
Planktothrix
variants. In several strains the gene variants seem to be a result of recombination. Both NRPS and non-NRPS genes are dominated by purifying selection; however, sites subjected to positive selection were also detected. The presence of similar and well-separated
Planktothrix
variants with low internal genetic diversity indicates gene flow within
Planktothrix
populations. Further, the low genetic diversity found between lakes (similar range as within lakes) indicates gene flow also between
Planktothrix
populations and suggests recent, or recurrent, dispersals. Our data also indicate that recombination has resulted in new genetic variants. Stability within variants and the development of new variants are likely to be influenced by selection patterns and within-variant homologous recombination.
Comparing Metabolic Functionalities, Community Structures, and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations
Authors: Gözdereliler Erkin , Boon Nico , Aamand Jens , De Roy Karen , Granitsiotis Michael S. , Albrechtsen Hans-Jørgen , Sørensen Sebastian R.
Published: November 2012

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter
−1
) or high (25 mg liter
−1
) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.
Molecular Control of Sucrose Utilization in Escherichia coli W, an Efficient Sucrose-Utilizing Strain
Authors: Sabri Suriana , Nielsen Lars K. , Vickers Claudia E.
Published: November 2012

Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in
Escherichia coli
, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (
csc
) genes in
E. coli
W were examined by knockout and overexpression experiments. At low sucrose concentrations, the
csc
genes are repressed and cells cannot grow. Removal of either the repressor protein (
cscR
) or the fructokinase (
cscK
) gene facilitated derepression. Furthermore, combinatorial knockout of
cscR
and
cscK
conferred an improved growth rate on low sucrose. The invertase (
cscA
) and sucrose transporter (
cscB
) genes are essential for sucrose catabolism in
E. coli
W, demonstrating that no other genes can provide sucrose transport or inversion activities. However,
cscK
is not essential for sucrose utilization. Fructose is excreted into the medium by the
cscK
-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of
cscA
,
cscAK
, or
cscAB
could complement the WΔ
cscRKAB
knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing
cscAB
, and full growth rate complementation in WΔ
cscRKAB
also required
cscAB
. Our understanding of sucrose utilization can be used to improve
E. coli
W and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.
Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor
Authors: Luo Xiaoxia , Chen Ling , Huang Qiong , Zheng Jinshui , Zhou Wei , Peng Donghai , Ruan Lifang , Sun Ming
Published: November 2012

Some
Bacillus thuringiensis
strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The
B. thuringiensis
strain YBT-1518 has three
cry
genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Gram-positive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against
Caenorhabditis elegans
(the 50% lethal concentration is 610 ± 9.37 μg/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against
C. elegans
. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the
B. thuringiensis
Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between
B. thuringiensis
and its host nematodes.
Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis
Authors: Ng Daphne T. W. , Sarkar Casim A.
Published: November 2012

Lactococcus lactis
is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from
L. lactis
, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of
Staphylococcus aureus
nuclease and further evaluated them for secretion of
Bacillus subtilis
α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered
L. lactis
signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.
Molecular Characterization of Human-Pathogenic Microsporidia and Cyclospora cayetanensis Isolated from Various Water Sources in Spain: a Year-Long Longitudinal Study
Authors: Galván Ana Luz , Magnet Angela , Izquierdo Fernando , Fenoy Soledad , Rueda Cristina , Fernández Vadillo Carmen , Henriques-Gil Nuno , del Aguila Carmen
Published: November 2012

Recent studies suggest the involvement of water in the epidemiology of
Cyclospora cayetanensis
and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223),
Cyclospora
spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including
Enterocytozoon bieneusi
(C, D, and D-like genotypes),
Encephalitozoon intestinalis
,
Encephalitozoon cuniculi
(genotypes I and III), and
Anncaliia algerae
.
C. cayetanensis
was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of
C. cayetanensis
in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.
Regulons of Three Pseudomonas syringae pv. tomato DC3000 Iron Starvation Sigma Factors
Authors: Markel Eric , Butcher Bronwyn G. , Myers Christopher R. , Stodghill Paul , Cartinhour Sam , Swingle Bryan
Published: November 2012

Pseudomonas syringae
pv. tomato DC3000 contains genes for 15 sigma factors. The majority are members of the extracytoplasmic function class of sigma factors, including five that belong to the iron starvation subgroup. In this study, we identified the genes controlled by three iron starvation sigma factors. Their regulons are composed of a small number of genes likely to be involved in iron uptake.
Construction of a Broad-Host-Range Tn
7
-Based Vector for Single-Copy P
BAD
-Controlled Gene Expression in Gram-Negative Bacteria
Authors: Damron F. Heath , McKenney Elizabeth S. , Schweizer Herbert P. , Goldberg Joanna B.
Published: November 2012

We describe a mini-Tn
7
-based broad-host-range expression cassette for arabinose-inducible gene expression from the P
BAD
promoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, using
Pseudomonas aeruginosa
as the model host.
Diet-Induced Alterations of Host Cholesterol Metabolism Are Likely To Affect the Gut Microbiota Composition in Hamsters
Authors: Martínez Inés , Perdicaro Diahann J. , Brown Andrew W. , Hammons Susan , Carden Trevor J. , Carr Timothy P. , Eskridge Kent M. , Walter Jens
Published: November 2012

The gastrointestinal microbiota affects the metabolism of the mammalian host and has consequences for health. However, the complexity of gut microbial communities and host metabolic pathways make functional connections difficult to unravel, especially in terms of causation. In this study, we have characterized the fecal microbiota of hamsters whose cholesterol metabolism was extensively modulated by the dietary addition of plant sterol esters (PSE). PSE intake induced dramatic shifts in the fecal microbiota, reducing several bacterial taxa within the families
Coriobacteriaceae
and
Erysipelotrichaceae
. The abundance of these taxa displayed remarkably high correlations with host cholesterol metabolites. Most importantly, the associations between several bacterial taxa with fecal and biliary cholesterol excretion showed an almost perfect fit to a sigmoidal nonlinear model of bacterial inhibition, suggesting that host cholesterol excretion can shape microbiota structure through the antibacterial action of cholesterol.
In vitro
experiments suggested a modest antibacterial effect of cholesterol, and especially of cholesteryl-linoleate, but not plant sterols when included in model bile micelles. The findings obtained in this study are relevant to our understanding of gut microbiota-host lipid metabolism interactions, as they provide the first evidence for a role of cholesterol excreted with the bile as a relevant host factor that modulates the gut microbiota. The findings further suggest that the connections between
Coriobacteriaceae
and
Erysipelotrichaceae
and host lipid metabolism, which have been observed in several studies, could be caused by a metabolic phenotype of the host (cholesterol excretion) affecting the gut microbiota.
Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity
Authors: Kautz Stefanie , Rubin Benjamin E. R. , Russell Jacob A. , Moreau Corrie S.
Published: November 2012

We are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle ant
Cephalotes varians
were
Rhizobiales
,
Burkholderiales
,
Opitutales
,
Xanthomonadales
, and
Campylobacterales
, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history of
Cephalotes
ants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with a
Spiroplasma
sp. (order
Entomoplasmatales
), a potential ant pathogen. Our work shows that the microbiome associated with
Cephalotes varians
is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity.
Circadian Activation of the Mitogen-Activated Protein Kinase MAK-1 Facilitates Rhythms in Clock-Controlled Genes in Neurospora crassa
Authors: Bennett Lindsay D. , Beremand Phillip , Thomas Terry L. , Bell-Pedersen Deborah
Published: November 2012

The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus
Neurospora crassa
. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in
Saccharomyces cerevisiae
and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from
Neurospora
cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ∼500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e.,
ccg-1
and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.
Normal Adaptation of Candida albicans to the Murine Gastrointestinal Tract Requires Efg1p-Dependent Regulation of Metabolic and Host Defense Genes
Authors: Pierce Jessica V. , Dignard Daniel , Whiteway Malcolm , Kumamoto Carol A.
Published: November 2012

Although gastrointestinal colonization by the opportunistic fungal pathogen
Candida albicans
is generally benign, severe systemic infections are thought to arise due to escape of commensal
C. albicans
from the gastrointestinal (GI) tract. The
C. albicans
transcription factor Efg1p is a major regulator of GI colonization, hyphal morphogenesis, and virulence. The goals of this study were to identify the Efg1p regulon during GI tract colonization and to compare
C. albicans
gene expression during colonization of different organs of the GI tract. Our results identified significant differences in gene expression between cells colonizing the cecum and ileum. During colonization,
efg1

−

null mutant cells expressed higher levels of genes involved in lipid catabolism, carnitine biosynthesis, and carnitine utilization than did colonizing wild-type (WT) cells. In addition, during laboratory growth,
efg1

−

null mutant cells grew to a higher density than WT cells. The
efg1

−

null mutant grew in depleted medium, while WT cells could grow only if the depleted medium was supplemented with carnitine, a compound that promotes the metabolism of fatty acids. Altered gene expression and altered growth capability support the ability of
efg1
−
cells to hypercolonize naïve mice. Also, Efg1p was shown to be important for transcriptional responses to the stresses present in the cecum environment. For example, during colonization,
SOD5
, encoding a superoxide dismutase, was highly upregulated in an Efg1p-dependent manner. Ectopic expression of
SOD5
in an
efg1

−

null mutant increased the fitness of the
efg1

−

null mutant cells during colonization. These data show that
EFG1
is an important regulator of GI colonization.
Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei
Authors: Sykes Steven E. , Hajduk Stephen L.
Published: November 2012

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.
Molecular and Morphological Data Support the Existence of a Sexual Cycle in Species of the Genus Paracoccidioides
Authors: Teixeira Marcus de Melo , Theodoro Raquel Cordeiro , Derengowski Lorena da Silveira , Nicola André Moraes , Bagagli Eduardo , Felipe Maria Sueli
Published: November 2012

The genus
Paracoccidioides
includes the thermodimorphic species
Paracoccidioides brasiliensis
and
P. lutzii
, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with
Paracoccidioides
species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus
Paracoccidioides
, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and
Saccharomyces cerevisiae
demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both
Paracoccidioides
species was also detected by real-time PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types.
In vitro
crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (
MAT1-1
) and the high-mobility-group (HMG) domain (
MAT1-2
), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus
Paracoccidioides
.
Missense Mutations in Cytochrome
c
Maturation Genes Provide New Insights into Rhodobacter capsulatus cbb
3
-Type Cytochrome
c
Oxidase Biogenesis
Authors: Ekici Seda , Jiang Xinpei , Koch Hans-Georg , Daldal Fevzi
Published: November 2012

The
Rhodobacter capsulatus cbb
3
-type cytochrome
c
oxidase (
cbb
3
-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the
ccoNOQP
operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (
ccoGHIS
). It also relies on the
c
-type cytochrome maturation (Ccm) process, which requires the
ccmABCDEFGHI
genes, because two of the
cbb
3
-Cox subunits (CcoO and CcoP) are
c
-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of
cbb
3
-Cox unless the growth medium is supplemented with copper. In this work, we isolated “Cu-unresponsive” derivatives of a
ccoA
deletion strain that exhibited no
cbb
3
-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the
ccmA
or
ccmF
gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of
cbb
3
-Cox. Subsequent construction and examination of single
ccm
knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the
ccm
knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes
c
1
and
c
2
than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various
c
-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of
R. capsulatus cbb
3
-Cox proceeds independently of that of the
c
-type cytochromes during the biogenesis of this enzyme.
Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth
Authors: Liu Jun , Hicks David B. , Krulwich Terry A.
Published: November 2012

AtpI, a membrane protein encoded by many bacterial
atp
operons, is reported to be necessary for
c
-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of
atpI
that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic
Bacillus pseudofirmus
OF4. A strain with a chromosomal deletion of
atpI
grew nonfermentatively, and its purified ATP synthase had a
c
-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of
atpI
, but not
atpZ
, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F
1
domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both
spoIIIJ
and
yqjG
, but not
atpI
or
atpZ
, complemented a YidC-depleted
Escherichia coli
strain. Consistent with such overlapping functions, single deletions of
spoIIIJ
or
yqjG
in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of
yqjG
was elevated at pH 7.5 relative to that at pH 10.5 and in Δ
spoIIIJ
strains, but it was lower than constitutive
spoIIIJ
expression. Deletion of
atpZ
caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.
Role of Homologous Recombination in Adaptive Diversification of Extraintestinal Escherichia coli
Authors: Paul Sandip , Linardopoulou Elena V. , Billig Mariya , Tchesnokova Veronika , Price Lance B. , Johnson James R. , Chattopadhyay Sujay , Sokurenko Evgeni V.
Published: November 2012

The contribution of homologous exchange (recombination) of core genes in the adaptive evolution of bacterial pathogens is not well understood. To investigate this, we analyzed fully assembled genomes of two
Escherichia coli
strains from sequence type 131 (ST131), a clonal group that is both the leading cause of extraintestinal
E. coli
infections and the main source of fluoroquinolone-resistant
E. coli
. Although the sequences of each of the seven multilocus sequence typing genes were identical in the two ST131 isolates, the strains diverged from one another by homologous recombination that affected at least 9% of core genes. This was on a par with the contribution to genomic diversity of horizontal gene transfer and point gene mutation. The genomic positions of recombinant and mobile genetic regions were partially linked, suggesting their concurrent occurrence. One of the genes affected by homologous recombination was
fimH
, which encodes mannose-specific type 1 fimbrial adhesin, resulting in functionally distinct copies of the gene in ST131 strains. One strain, a uropathogenic isolate, had a pathoadaptive variant of
fimH
that was acquired by homologous replacement into the commensal strain background. Close examination of FimH structure and function in additional ST131 isolates revealed that recombination led to acquisition of several functionally distinct variants that, upon homologous exchange, were targeted by a variety of pathoadaptive mutations under strong positive selection. Different recombinant
fimH
strains also showed a strong clonal association with ST131 isolates that had distinct fluoroquinolone resistance profiles. Thus, homologous recombination of core genes plays a significant role in adaptive diversification of bacterial pathogens, especially at the level of clonally related groups of isolates.