bluuebunny/crossref_metadata_2025 huggingface dataset:
The Low Load Limit of Gasoline Partially Premixed Combustion Using Negative Valve Overlap
Authors: Borgqvist Patrick , Andersson Öivind , Tunestål Per , Johansson Bengt
Published: July 2013

Partially premixed combustion has the potential of high efficiency and simultaneous low soot and NOx emissions. Running the engine in partially premixed combustion mode with high octane number fuels has the advantage of a longer premix period of fuel and air which reduces soot emissions, even at higher loads. The problem is the ignitability at low load and idle operating conditions. The objective is to investigate different multiple-injection strategies in order to further expand the low load limit and reduce the dependency on negative valve overlap in order to increase efficiency. The question is, what is the minimum attainable load for a given setting of negative valve overlap and fuel injection strategy. The experimental engine is a light duty diesel engine equipped with a fully flexible valve train system. The engine is run without boost at engine speed 800 rpm. The fuel is 87 RON gasoline. A turbocharger is typically used to increase the boost pressure, but at low engine speed and load the available boost is expected to be limited. The in-cylinder pressure and temperature around top-dead-center will then be too low to ignite high octane number fuels. A negative valve overlap can be used to extend the low engine speed and load operating region. But one of the problems with negative valve overlap is the decrease in gas-exchange efficiency due to heat-losses from recompression of the residual gases. Also, the potential temperature increase from the trapped hot residual gases is limited at low load due to the low exhaust gas temperature. In order to expand the low load operating region further, more advanced injection strategies are investigated.
Combined Experimental and Computational Investigation of Unsteady Structure of Sheet/Cloud Cavitation
Authors: Huang Biao , Young Yin L. , Wang Guoyu , Shyy Wei
Published: July 2013

The objective of this paper is to apply combined experimental and computational modeling to investigate unsteady sheet/cloud cavitating flows. In the numerical simulations, a filter-based density corrected model (FBDCM) is introduced to regulate the turbulent eddy viscosity in both the cavitation regions on the foil and in the wake, which is shown to be critical in accurately capturing the unsteady cavity shedding process, and the corresponding velocity and vorticity dynamics. In the experiments, high-speed video and particle image velocimetry (PIV) technique are used to measure the flow velocity and vorticity fields, as well as cavitation patterns. Results are presented for a Clark-Y hydrofoil fixed at an angle of attack of α = 8 deg at a moderate Reynolds number, Re = 7 × 105, for both subcavitating and sheet/cloud cavitating conditions. The results show that for the unsteady sheet/cloud cavitating case, the formation, breakup, shedding, and collapse of the sheet/cloud cavity lead to substantial increase in turbulent velocity fluctuations in the cavitating region around the foil and in the wake, and significantly modified the wake patterns. The turbulent boundary layer thickness is found to be much thicker, and the turbulent intensities are much higher in the sheet/cloud cavitating case. Compared to the wetted case, the wake region becomes much broader and is directed toward the suction side instead of the pressure side for the sheet/cloud cavitation case. The periodic formation, breakup, shedding, and collapse of the sheet/cloud cavities, and the associated baroclinic and viscoclinic torques, are shown to be important mechanisms for vorticity production and modification.
Centrifugal Casting of Microfluidic Components With PDMS
Authors: Mazzeo Aaron D. , Hardt David E.
Published: July 2013

This work describes the centrifugal casting and fast curing of double-sided, polydimethylsiloxane (PDMS)-based components with microfeatures. Centrifugal casting permits simultaneous patterning of multiple sides of a component and allows control of the thickness of the part in an enclosed mold without entrapment of bubbles. Spinning molds filled with PDMS at thousands of revolutions per minute for several minutes causes entrapped bubbles within the PDMS to migrate toward the axis of rotation or dissolve into solution. To cure the parts quickly (<10 min), active elements heat and cool cavities filled with PDMS after the completion of spinning. Microfluidic channels produced from the process have a low coefficient of variation (<2% for the height and width of channels measured in 20 parts). This process is also capable of molding functional channels in opposite sides of a part as demonstrated through a device with a system of valves typical to multilayer soft lithography.
Nonlinear Dynamic Behavior of a Flexible Shaft Supported by Smart Hydrostatic Squeeze Film Dampers
Authors: Bouzidane A. , Thomas M.
Published: July 2013

The aim of this research is to study the nonlinear dynamic behavior of a flexible shaft supported by smart hydrostatic squeeze film dampers, which are filled with a negative electrorheological fluid (NERF). A nonlinear model of the hydrostatic squeeze film damper has been developed in order to study the effect of the electrorheological fluid on the dynamic behavior of a flexible shaft. The results obtained are discussed and compared with the linear model, which is restricted to only small vibrations around the equilibrium position. A new smart hydrostatic squeeze film damper is proposed to reduce the transient response of the shaft and transmitted forces by applying an electric field to the NER fluid, which results in modifying its viscosity. The results show that it is possible to effectively monitor the electric field and the viscosity of the fluid inside the hydrostatic squeeze film dampers (HSFD) for a better control of flexible shaft vibration and bearing transmitted forces.
Recent Advances of Internal Cooling Techniques for Gas Turbine Airfoils
Authors: Chyu Minking K. , Siw Sin Chien
Published: July 2013

The performance goal of modern gas turbine engines, both land-base and air-breathing engines, can be achieved by increasing the turbine inlet temperature (TIT). The level of TIT in the near future can reach as high as 1700 °C for utility turbines and over 1900 °C for advanced military engines. Advanced and innovative cooling techniques become one of the crucial major elements supporting the development of modern gas turbines, both land-based and air-breathing engines with continual increment of turbine inlet temperature (TIT) in order to meet higher energy demand and efficiency. This paper discusses state-of-the-art airfoil cooling techniques that are mainly applicable in the mainbody and trailing edge section of turbine airfoil. Potential internal cooling designs for near-term applications based on current manufacturing capabilities are identified. A literature survey focusing primarily on the past four to five years has also been performed.
Fault Diagnosis of a Sensor Network: A Distributed Filtering Approach
Authors: Doraiswami Rajamani , Cheded Lahouari
Published: July 2013

This paper proposes a model-based approach to develop a novel fault diagnosis scheme for a sensor network of a cascade, parallel and feedback combination of subsystems. The objective is to detect and isolate a fault in any of the subsystems and measurement sensors which are subject to disturbances and/or measurement noise. Our approach hinges on the use of a bank of Kalman filters (KF) to detect and isolate faults. Each KF is driven by either a pair (a) of consecutive sensor measurements or (b) of a reference input and a measurement. It is shown that the KF residual is a reliable indicator of a fault in subsystems and sensors located in the path between the pair of the KF's input. The simple and efficient procedure proposed here analyzes each of the associated paths and leads to both the detection and isolation of any fault that occurred in the paths analyzed. The scheme is successfully evaluated on several simulated examples and on a physical fluid system exemplified by a benchmarked laboratory-scale two-tank system to detect and isolate faults including sensor, actuator and leakage ones.
Observer-Based H∞ Control on Nonhomogeneous Discrete-Time Markov Jump Systems
Authors: Yin Yanyan , Shi Peng , Liu Fei , Teo Kok Lay
Published: July 2013

This paper concerns the problem of observer-based H∞ controller design for a class of discrete-time Markov jump systems with nonhomogeneous jump parameters. A nonhomogeneous jump transition probability matrix is described by a polytope set, in which values of vertices are given. By Lyapunov function approach, under the designed observer-based controller, a sufficient condition is presented to ensure the resulting closed-loop system is stochastically stable and a prescribed H∞ performance is achieved. Finally, a simulation example is given to show the effectiveness of the developed techniques.
An RFID-Driven Graphical Formalized Deduction for Describing the Time-Sensitive State and Position Changes of Work-in-Progress Material Flows in a Job-Shop Floor
Authors: Jiang Pingyu , Cao Wei
Published: July 2013

As a key advanced manufacturing technology in next generation manufacturing systems, radio frequency identification (RFID) technology is considered to be one of the most promising technological innovations with the potential to increase visibility and improve efficiency. Therefore, research about RFID and its applications are increasing by blasting with all kinds of RFID models in various fields, especially in manufacturing. By introducing RFID technology into the job-shop floor, this paper proposes a systematic RFID-driven graphical formalized deduction model (rfid-GFDM) for describing the time-sensitive state and position changes of work-in-progress (WIP) material flows and guiding where to deploy RFID devices and how to use them for collecting real-time on-site data. Four steps including RFID configuration based on the process flow model, state blocks model, automatic event generation, and extended event-driven model are proposed one by one to support the implementation of rfid-GFDM. The nature of RFID technology is revealed, too. A use case about a computer numerical control (CNC) milling system is studied, and it demonstrates the feasibility of the proposed model. Finally, the possibility of popularizing the model to other field is discussed, too. It is expected to establish a normative RFID modeling method that will facilitate the convenience of RFID applications in a broad scope.
Chatter Stability Model of Micro-Milling With Process Damping
Authors: Jin Xiaoliang , Altintas Yusuf
Published: July 2013

This paper presents the prediction of cutting forces and chatter stability of micro-milling operations from the material's constitutive flow stress and structural dynamics of the micro-end mill. The cutting force coefficients are identified either using previously presented slip-line field or finite element methods by considering the effects of chip size, cutting edge radius, rake angle and cutting speed. The process damping caused by the plowing of round edge is modeled by finite element method. The frequency response function of the fragile micro-mill is measured through specially devised piezo actuator mechanism. Dynamic model of micro-milling with the velocity dependent process damping mechanism is presented, and the chatter stability is predicted in frequency domain. The proposed models have been experimentally verified in micro-milling of AISI 1045 steel.
Droplet Detachment Mechanism in a High-Speed Gaseous Microflow
Authors: Carroll Brian , Hidrovo Carlos
Published: July 2013

This paper experimentally investigates the mechanism of water droplet detachment in a confined microchannel under highly inertial (10 < Re < 200) air flow conditions. Experimental observations show that as the Reynolds number of the continuous phase is increased, the droplet transitions from an elongated slug to a nearly uniform aspect ratio droplet. Supporting scaling arguments are then made that examine the relevant forces induced by the continuous phase on the droplet at the point of detachment. The inertial, viscous, and hydrodynamic pressure forces that result as the air flow is confined in the small gap between droplet and channel walls are compared to the surface tension force pinning the droplet at the injection site. The results indicate that the dominant detachment mechanism transitions from the hydrostatic pressure difference to inertial drag as the continuous phase velocity is increased.
Effect of Process Parameters on Burrs Produced in Micromilling of a Thin Nitinol Foil
Authors: Mathai George K. , Melkote Shreyes N. , Rosen David W.
Published: July 2013

This paper examines the formation of burrs in micromilling of a thin nickel–titanium alloy (nitinol) foil used in implantable biomedical device applications. The paper analyzes the effects of key micromilling process parameters such as spindle speed, feed, tool wear, backing material, and adhesive used to attach the foil to the backing material on the burr height. It is found that burr height is larger on the downmilling side for grooves cut with a worn tool at high feeds, low spindle speeds with a softer backing material, and a weaker adhesive bond. Some important interaction effects of these factors are also studied. The study also shows that the mechanics of burr formation in such thin materials depends on whether the mode of cutting is dominated by tearing or chip formation, which is a function of the feed rate. A kinematic model to predict burr widths is developed and verified through experiments.
Numerical Simulation of Coal Fly-Ash Erosion in an Induced Draft
Fan
Authors: Corsini Alessandro , Rispoli Franco , Sheard Anthony G. , Venturini Paolo
Published: July 2013

Induced draft fans extract coal-fired boiler exhaust gases in the form of a two-phase flow with a dispersed solid phase made of unburnt coal and fly ash; consequently fan blades are subject to erosion causing material wear at the leading edge, trailing edge, and blade surface. Erosion results in blade material loss, a reduction of blade chord, and effective camber that together degrade aerodynamic performance. This paper presents a numerical study of the erosive process in an induced draft fan carried out by simulating the particle laden flow using an original finite element Eulerian-Lagrangian solver. The particle trajectories are calculated using a particle cloud tracking technique that considers drifting near wall and an algebraic erosion model. The numerical study clarifies the influence of fan operation to the determination of the erosion regimes and patterns. In particular, the study investigates the role played by the size and mass distribution of the particles by considering a real composition of the flying ashes in the exhaust flow from a coal-fired boiler. The results illustrate the critical blade areas and erosion rates as given by the particle dynamics of different sizes. A specific analysis of the material wear at the blade leading edge is also given.
Mixing Evaluation of a Passive Scaled-Up Serpentine Micromixer With Slanted Grooves
Authors: Cook Kristina J. , Fan YanFeng , Hassan Ibrahim
Published: July 2013

A novel, passive, scaled-up micromixer based on fluid rotation is proposed and evaluated experimentally and numerically over Reynolds numbers ranging from 0.5 to 100. Flow visualization is employed to qualitatively assess flow patterns, while induced fluorescence is used to quantify species distribution at five locations along the channel length. Two individual fluids are supplied to the test section via a Y-inlet. The fluid enters a meandering channel with four semicircular portions, each of which is lined with nine slanted grooves at the bottom surface. The main mixing channel is 3 mm wide and 0.75 mm deep, with a total length of 155.8 mm. Numerical simulations confirm rotation at all investigated Reynolds numbers, and the strength of rotation increases with increasing Reynolds number. Grooves are employed to promote helical flow, while the serpentine channel structure results in the formation of Dean vortices at Re ≥ 50 (Dean number ≥ 18.25), where momentum has a more significant effect. A decreasing-increasing trend in the degree of mixing was noted, with an inflection point at Re = 5, marking the transition from diffusion dominance to advection dominance. The increase in interfacial surface area is credited with the improved mixing in the advection-dominant regime, while high residence time allowed for significant mass diffusion in the diffusion-dominant regime. Good mixing was achieved at both high and low Reynolds numbers, with a maximum mixing index of 0.90 at Re = 100.
Laser Welding of AISI 316 Steel: Microstructural and Stress Analysis
Authors: Yilbas B. S. , Akhtar Sohail
Published: July 2013

Thermal-stress field in the welded region was modeled incorporating the finite element model. Temperature and stress fields were predicted at different cooling periods. The morphological and metallurgical changes in the welded region were examined using optical and scanning electron microscopes, energy dispersive spectroscopy and X-ray diffraction. The residual stress formed at the surface vicinity of the weld was determined using the X-ray diffraction technique. It was found that the residual stress predicted agreed well with the experimental data. The solidification cracking did not occur in the weld section during the cooling period. The microhardness in the weld cross-section was almost 1.4 times the base material hardness.
Mechanics Interpretation on the Bending Stiffness and Wrinkled Pattern of Graphene
Authors: Xu Ran , Wang Yu , Liu Bin , Fang Daining
Published: July 2013

In this paper we attempt to answer two questions on graphene from a mechanic's viewpoint: why does this one-atom-thick monolayer have finite bending stiffness to ensure its stability? and what is its wrinkle mechanism? As for the first question, it is found that the repulsive residual internal moment in the bond angle can lead to a nonzero bending stiffness, which makes the graphene flat. Together with long-range attraction among atoms, such as van der Waals forces, a graphene prefers to have a self-buckling wrinkled configuration with many waves.
Uniaxial Tension of a Class of Compressible Solids With Plastic
Non-Normality
Authors: Mohan Nisha , Cheng Justine , Greer Julia R. , Needleman Alan
Published: July 2013

Motivated by a model that qualitatively captured the response of vertically aligned carbon nanotube (VACNT) pillars in uniaxial compression, we consider the uniaxial tensile response of a class of compressible elastic-viscoplastic solids. In Hutchens et al. [“Analysis of Uniaxial Compression of Vertically Aligned Carbon Nanotubes,” J. Mech. Phys. Solids, 59, pp. 2227–2237 (2011), Erratum 60, 1753–1756 (2012)] an elastic viscoplastic constitutive relation with plastic compressibility, plastic non-normality, and a hardening-softening-hardening hardness function was used to model experimentally obtained uniaxial compression data of cylindrical VACNT micropillars. Complex deformation modes were found in uniaxial compression, which include a sequential buckling-like collapse of the type seen in experiments. These complex deformation modes led to the overall stress-strain signature of the pillar not being of the same form as the input material hardness function. A fundamental question that motivates exploring the deformation of this class of materials—both experimentally and theoretically—is how to extract the intrinsic material response from simple tests. In this study we explore the relation between the input material response and the overall stress strain behavior in uniaxial tension using the constitutive framework of Hutchens et al. A simple one-dimensional analysis reveals the types of instability modes to be expected. Dynamic, finite deformation finite element calculations are carried out to explore the dependence of diffuse necking, localized necking, and propagating band deformation modes on characteristics of the hardness function. Attention is devoted to uncovering implications for obtaining intrinsic material properties of complex hierarchical structures; for example, vertically aligned carbon nanotubes (VACNTs), from uniaxial tension experiments.
Effect of Finite Diaphragm Rupture Process on Microshock Tube Flows
Authors: K. R Arun , Kim H. D. , Setoguchi T.
Published: July 2013

The study of flow physics in microshock tubes is of growing importance with the recent development of microscale technology. The flow characteristics in a microshock tube is considerably different from that of the conventional macroshock tube due to the boundary layer effects and high Knudsen number effects. In the present study an axisymmetric computational fluid dynamics (CFD) method was employed to simulate the microshock tube flow field with Maxwell's slip velocity and temperature jump boundary conditions, to accommodate the rarefaction effects. The effects of finite diaphragm rupture process and partial diaphragm rupture on the flow field and the wave propagations were investigated, in detail. The results show that the shock propagation distance attenuates rapidly for a microshock tube compared to a macroshock tube. For microshock tubes, the contact surface comes closer to the shock front compared to the analytical macroshock tube case. Due to the finite diaphragm rupture process the moving shock front will be generated after a certain distance ahead of the diaphragm and get attenuated rapidly as it propagates compared to the sudden rupture case. The shock-contact distance reduces considerably for the finite diaphragm rupture case compared to the sudden diaphragm rupture process. A partially burst diaphragm within a microshock tube initiates a supersonic flow in the vicinity of the diaphragm similar to that of a supersonic nozzle flow. The supersonic flow expansion leads to the formation of oblique shock cells ahead of the diaphragm and significantly attenuates the moving shock propagation speed.
Numerical Simulation of Turbulent Mist Flows With Liquid Film Formation in Curved Pipes Using an Eulerian–Eulerian Method
Authors: Zhang Pusheng , Roberts Randy M. , Bénard André
Published: July 2013

Turbulent flows of air/water mixtures through curved pipes are modeled in this work using a Eulerian–Eulerian method. This is motivated by the possibility of using computational fluid dynamics (CFD) as a design tool applied to curved pipes feeding a gas/liquid separator. The question is to identify the curvature of such pipes that can promote film formation upstream of the separator and, thus, precondition the flow without creating a large pressure drop. The performance of the mixture theory with a drift flux model and the “realizable” k-ε closure was evaluated in the simulations. The enhanced wall treatment (EWT) was utilized to resolve the flow in the near-wall region. A qualitative study was first conducted to investigate the flow patterns and the liquid film formation in a 180 deg bend. The numerical results were validated by comparing the computed pressure drop with empirical correlations from the literature. Subsequently, the importance of droplet size and liquid volume fraction was investigated by studying their effect on the flow patterns of the continuous phase, as well as their impact on the secondary flow intensity, the pressure drop, and the liquid film formation on the wall. Various pipe geometries were studied to achieve a low pressure drop while maintaining a high droplet deposition. Results show that a combination of the drift flux model with the realizable k-ε closure and EWT for the near-wall treatment appears capable of capturing the complex secondary flow patterns such as those associated with film inversion. The pressure drop computed for various flows appear to be in good agreement with an empirical correlation. Finally, bends with a curvature ratio around 7 appear to be the optimal for providing a small pressure drop as well as a high droplet deposition efficiency in a U-bend.
Analysis and Design of Wire Transport System in Microwire-Electronic Discharge Machining
Authors: Wang P. W. , Yang C. S.
Published: July 2013

A dynamic model for analyzing the wire transport system of micro w-EDM (wire electronic discharge machining) is proposed. Based on the model, two mechanisms are proposed to stabilize the wire tension. The first mechanism is the active wire feed apparatus where the wire spool is fed by a motor actively, instead of passively pulled by the windup motor. Hence, the inertia loading of the wire spool can be isolated from the system. The second mechanism is mounting a multilayer damped vibration absorber (MDVA) on the system. As the wire tension variation occurs, the MDVA oscillates to attenuate the wire tension variation. The performances of both mechanisms on the wire tension variation are theoretically investigated and experimentally validated through corner cutting on the 1.0 mm thickness tungsten carbide. Results show that the wire tension variation can be reduced from 10.3 gf to 3.3 gf after mounting the active wire feed apparatus and the oscillation frequency is increased from 13 Hz to 21 Hz. The wire tension variation can be further reduced to 1.9 gf after mounting the MDVA on the system and the high frequency perturbation is significantly attenuated. The 30-deg corner cutting shows that the corner error are significantly reduced from 26.0 μm to 12.0 μm; the standard deviation of kerf is reduced from 4.34 μm to 0.96 μm, and the surface roughness Ra is reduced from 1.15 μm to 0.63 μm after employing both developed mechanisms.
Efficiency of High-Sensitivity Gear Trains, Such as Cycloid Drives
Authors: Sensinger Jonathon W.
Published: July 2013

Compact, high torque ratio, high efficiency transmissions are required in many applications, such as robotics. However, compact size and high torque ratios often come at the expense of surprisingly low efficiency. Here we apply Del Castillo's sensitivity framework (Del Castillo, J. M., 2002, “The Analytical Expression of the Efficiency of Planetary Gear Trains,” Mech. Mach. Theory, 37(2), pp. 197–214) to explain the low efficiency of a subset of transmissions that exploit small differences in tooth number between gears to generate high torque ratios. We add adjustment factors for several transmissions within this category, such as cycloids and harmonic drives; demonstrate that the models match empirical results for the case of cycloids across a range of torque ratios, topologies, and roller conditions; and compare and optimize the topologies of the various mechanisms. We demonstrate that for this subset of transmissions, the efficiency approaches a function of the torque ratio.
Numerical Simulations for Hydrodynamics of Air-Water External Loop Airlift Reactor Flows With Bubble Break-Up and Coalescence Effects
Authors: Law Deify , Battaglia Francine
Published: July 2013

The external loop airlift reactor (ELALR) is a modified bubble column reactor that is composed of two vertical columns interconnected with two horizontal tubes and is often preferred over traditional bubble column reactors because it can operate over a wider range of conditions. In the present work, the gas-liquid flow dynamics in an ELALR were simulated using an Eulerian–Eulerian ensemble-averaging method with bubble breakup and coalescence effects in a three-dimensional system. The population balance models (PBM) of Luo and Svendsen (1996, “Theoretical Model for Drop and Bubble Breakup in Turbulent Dispersions,” AIChE J., 42, pp. 1225–1233) and Prince and Blanch (1990, “Bubble Coalescence and Breakup in Air-Sparged Bubble Columns,” AIChE J., 36, pp. 1485–1499) were used to simulate the bubble breakup and coalescence effects, respectively. The bubble breakup and coalescence closure models were implemented into CFDLib, a multiphase flow source code developed by Los Alamos National Laboratory, and validated with experiments. The computational fluid dynamics (CFD) simulations were then compared to experimental measurements from a 10.2 cm diameter ELALR for superficial gas velocities ranging from 1 to 20 cm/s. From this work, the 3D PBM simulations of an external loop airlift reactor were generally comparable with the 3D single bubble size simulations. However, the 3D PBM simulations have closer agreement with experimental findings than the single bubble size simulations especially regarding the length of gas bubbles in the downcomer.
Nucleation and growth of MgO atomic layer deposition: A real-time spectroscopic ellipsometry study
Authors: Wang Han , Fu Kan
Published: July 2013

The atomic layer deposition (ALD) of MgO thin films from bis(cyclopentadienyl) magnesium and H2O was studied using in-situ real-time spectroscopic ellipsometry (SE), ex-situ x-ray photoelectron spectroscopy, and grazing-incidence x-ray diffraction. It is found that the initial growth is not linear during the first ten cycles, and magnesium silicate forms spontaneously on the SiO2/Si substrates at 250 °C. Submonolayer sensitivity of SE is demonstrated by the analysis of each half-cycle and self-limiting adsorption, revealing characteristic features of hetero- and homo-MgO ALD processes.
Evolution of Pseudomonas aeruginosa Virulence as a Result of Phage Predation
Authors: Hosseinidoust Zeinab , van de Ven Theo G. M. , Tufenkji Nathalie
Published: July 2013

The rapid increase in the emergence of antibiotic-resistant bacteria has attracted attention to bacteriophages for treating and preventing bacterial infections. Bacteriophages can drive the diversification of
Pseudomonas aeruginosa
, giving rise to phage-resistant variants with different phenotypes from their ancestral hosts. In this study, we sought to investigate the effect of phage resistance on cytotoxicity of host populations toward cultured mammalian cells. The library of phage-resistant
P. aeruginosa
PAO1 variants used was developed previously via experimental evolution of an isogenic host population using phages PP7 and E79. Our results presented herein indicate that the phage-resistant variants developed in a heterogeneous phage environment exhibit a greater ability to impede metabolic action of cultured human keratinocytes and have a greater tendency to cause membrane damage even though they cannot invade the cells in large numbers. They also show a heightened resistance to phagocytosis by model murine macrophages. Furthermore, all isolates produced higher levels of at least one of the secreted virulence factors, namely, total proteases, elastase, phospholipase C, and hemolysins. Reverse transcription-quantitative PCR (RT-qPCR) revealed upregulation in the transcription of a number of genes associated with virulence of
P. aeruginosa
for the phage-resistant variants. The results of this study indicate a significant change in the
in vitro
virulence of
P. aeruginosa
following phage predation and highlight the need for caution in the selection and design of phages and phage cocktails for therapeutic use.
Comparison of Listeria monocytogenes Exoproteomes from Biofilm and Planktonic State: Lmo2504, a Protein Associated with Biofilms
Authors: Lourenço António , de Las Heras Aitor , Scortti Mariela , Vazquez-Boland Jose , Frank Joseph F. , Brito Luisa
Published: July 2013

The food-borne pathogen
Listeria monocytogenes
is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δ
lmo2504
), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δ
lmo2504
showed a significantly (
P
< 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.
An Extracellular Serine/Threonine-Rich Protein from Lactobacillus plantarum NCIMB 8826 Is a Novel Aggregation-Promoting Factor with Affinity to Mucin
Authors: Hevia Arancha , Martínez Noelia , Ladero Víctor , Álvarez Miguel A. , Margolles Abelardo , Sánchez Borja
Published: July 2013

Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in
Lactobacillus plantarum
. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in
Lactococcus lactis
. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in
Lb. plantarum
.
Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli
Authors: Furuya Toshiki , Hayashi Mika , Kino Kuniki
Published: July 2013

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly inEscherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD inEscherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of themimABCDgene expression inE. colirevealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction ofE. colicells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformedE. colicells inactive. We found that the following factors are important for functional expression of MimABCD inE. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble inE. colicells, and the optimization of themimDnucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed inE. coli. The strategy described here should be generally applicable to theE. coliexpression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.
Characterization of Pleurotus ostreatus Biofilms by Using the Calgary Biofilm Device
Authors: Pesciaroli Lorena , Petruccioli Maurizio , Fedi Stefano , Firrincieli Andrea , Federici Federico , D'Annibale Alessandro
Published: July 2013

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of
Pleurotus ostreatus
biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of
P. ostreatus
biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts.
Posttranslational Maturation of the Invasion Acyl Carrier Protein of Salmonella enterica Serovar Typhimurium Requires an Essential Phosphopantetheinyl Transferase of the Fatty Acid Biosynthesis Pathway
Authors: Viala Julie P. M. , Puppo Rémy , My Lætitia , Bouveret Emmanuelle
Published: July 2013

Salmonella
pathogenicity island 1 (SPI-1) carries genes required for the formation of a type 3 secretion system, which is necessary for the invasion process of
Salmonella
. Among the proteins encoded by SPI-1 is IacP, a homolog of acyl carrier proteins. Acyl carrier proteins are mainly involved in fatty acid biosynthesis, and they require posttranslational maturation by addition of a 4′-phosphopantetheine prosthetic group to be functional. In this study, we analyzed IacP maturation
in vivo
. By performing matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry analysis of intact purified proteins, we showed that IacP from
Salmonella enterica
serovar Typhimurium was matured by addition of 4′-phosphopantetheine to the conserved serine 38 residue. Therefore, we searched for the phosphopantetheinyl transferases in charge of IacP maturation. A bacterial two-hybrid approach revealed that IacP interacted with AcpS, an enzyme normally required for the maturation of the canonical acyl carrier protein (ACP), which is involved in fatty acid biosynthesis. The creation of a conditional
acpS
mutant then demonstrated that AcpS was necessary for the maturation of IacP. However, although IacP was similar to ACP and matured by using the same enzyme, IacP could not replace the essential function of ACP in fatty acid synthesis. Hence, the demonstration that IacP is matured by AcpS establishes a cross-connection between virulence and fatty acid biosynthesis pathways.
Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis
Authors: Fung Danny Ka Chun , Lau Wai Yin , Chan Wing Tat , Yan Aixin
Published: July 2013

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting
Escherichia coli
to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in
E. coli
without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P

cusC

-
lacZ
reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of
E. coli
, resulting in Cu(I) toxicity. Cells lacking
cusCFBA
and another copper transporter,
copA
, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of Δ
cusC
cells. Yet, Cu(I) treatment did not impair the Frd activity
in vitro
. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and Δ
cusC
enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of
E. coli
.
Identification of a Ligand on the Wip1 Bacteriophage Highly Specific for a Receptor on Bacillus anthracis
Authors: Kan Sherry , Fornelos Nadine , Schuch Raymond , Fischetti Vincent A.
Published: July 2013

Tectiviridae
is a family of tailless bacteriophages with Gram-negative and Gram-positive hosts. The family model PRD1 and its close relatives all infect a broad range of enterobacteria by recognizing a plasmid-encoded conjugal transfer complex as a receptor. In contrast, tectiviruses with Gram-positive hosts are highly specific to only a few hosts within the same bacterial species. The cellular determinants that account for the observed specificity remain unknown. Here we present the genome sequence of Wip1, a tectivirus that infects the pathogen
Bacillus anthracis
. The Wip1 genome is related to other tectiviruses with Gram-positive hosts, notably, AP50, but displays some interesting differences in its genome organization. We identified Wip1 candidate genes for the viral spike complex, the structure located at the capsid vertices and involved in host receptor binding. Phage adsorption and inhibition tests were combined with immunofluorescence microscopy to show that the Wip1 gene product p23 is a receptor binding protein. His-p23 also formed a stable complex with p24, a Wip1 protein of unknown function, suggesting that the latter is involved with p23 in host cell recognition. The narrow host range of phage Wip1 and the identification of p23 as a receptor binding protein offer a new range of suitable tools for the rapid identification of
B. anthracis
.
NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO
Authors: Wang Shuning , Huang Haiyan , Kahnt Jörg , Mueller Alexander P. , Köpke Michael , Thauer Rudolf K.
Published: July 2013

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic
Clostridium autoethanogenum
autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (
fdhA/hytA-E
) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP
+
with H
2
or formate and the reversible formation of H
2
and CO
2
from formate. We propose the complex to have two functions
in vivo
, namely, to normally catalyze CO
2
reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H
2
formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of
C. autoethanogenum
on CO (
E
0′
= −520 mV).
Characterization of SAV7471, a TetR-Family Transcriptional Regulator Involved in the Regulation of Coenzyme A Metabolism in Streptomyces avermitilis
Authors: Liu Yanping , Yan Tingting , Jiang Libin , Wen Ying , Song Yuan , Chen Zhi , Li Jilun
Published: July 2013

The role of a
tetR
transcriptional regulatory gene (
SAV7471
) in avermectin production in the Gram-positive soil bacterium
Streptomyces avermitilis
was investigated by gene deletion, complementation, and overexpression experiments. Gene deletion of the
SAV7471
open reading frame resulted in avermectin overproduction. The deletion also resulted in overexpression of
SAV7472-SAV7473
transcripts, which encode a protein of unknown function and a flavoprotein possibly involved in pantothenate and coenzyme A (CoA) metabolism. EMSAs and footprinting assays showed that SAV7471 can bind to two palindromic sequences with high similarity in the intergenic region between
SAV7471
and
SAV7472
, a region that contains the apparent transcription start sites for each gene detected by rapid amplification of 5′ cDNA ends (5′-RACE). In addition to
SAV7472-SAV7473
, at least two genes (
SAV1104
and
SAV1258
) involved in CoA metabolism are negatively controlled by SAV7471. By negatively regulating the transcription of the target genes
SAV7472-SAV7473
and other genes involved in CoA metabolism, SAV7471 may affect cellular metabolic flux and may thereby indirectly regulate avermectin biosynthesis.