bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal Characteristics of Gaseous Fuel Flames Using High Temperature Air
Authors: Gupta A. K.
Published: March 2004

Recent advances on high temperature air combustion (HiTAC) have demonstrated significant energy savings, higher and uniform thermal field, lower pollution, and smaller size of the equipment for a range of furnace applications. The HiTAC technology has evolved from the conception of excess enthalpy combustion (EEC) to high and ultra-high preheated air combustion. In the HiTAC method, combined heat regeneration and low oxygen methods are utilized to enlarge and control the flame thermal behavior. This technology has shown promise for much wider applications in various process and power industries, energy conversion, and waste to clean fuel conversion. For each application the flow, thermal, and chemical behavior of HiTAC flames must be carefully tailored to satisfy the specific needs. Qualitative and quantitative results are presented on several gas-air diffusion flames using high-temperature combustion air. A specially designed regenerative combustion test furnace facility, built by Nippon Furnace Kogyo, Japan, was used to preheat the combustion air to elevated temperatures. The flames with highly preheated combustion air were significantly more stable and homogeneous (both temporally and spatially) as compared to the flames with room-temperature combustion air. The global flame features showed the flame color to change from yellow to blue to bluish-green to green over the range of conditions examined. In some cases hybrid and purple color flame was also observed. Under certain conditions flameless or colorless oxidation of the fuel has also been demonstrated. Information on global flame features, flame spectral emission characteristics, spatial distribution of OH, CH, and C2 species and emission of pollutants has been obtained. Low levels of NOx along with negligible levels of CO and HC have been obtained using high-temperature combustion air. The thermal and chemical behavior of high-temperature air combustion flames depends on fuel property, preheat temperature, and oxygen concentration of air. Waste heat from a furnace in high-temperature air combustion technology is retrieved and introduced back into the furnace using regenerator. These features help save energy, which subsequently also reduce the emission of CO2 (greenhouse gas) to the environment. Flames with high temperature air provide significantly higher and uniform heat flux than normal air, which reduces the equipment size or increases the process material throughput for same size of the equipment. The high-temperature air combustion technology can provide significant energy savings (up to about 60%), downsizing of the equipment (about 30%), and pollution reduction (about 25%). Fuel energy savings directly translates to a reduction of CO2 and other greenhouse gases to the environment.
Humid Air NOx Reduction Effect on Liquid Fuel Combustion
Authors: Chen A. G. , Maloney Daniel J. , Day William H.
Published: March 2004

An experimental investigation was carried out at DOE NETL on the humid air combustion process using liquid fuel to determine the effects of humidity on pollutant emissions and flame stability. Tests were conducted at pressures of up to 100 psia (690 kPa), and a typical inlet air temperature of 860°F (733 K). The emissions and RMS pressures were documented for a relatively wide range of flame temperature from 2440-3090°F (1610–1970 K) with and without added humidity. The results show more than 90% reduction of NOx through 10% humidity addition to the compressed air compared with the dry case at the same flame temperature. The substantial reduction of NOx is due to a shift in the chemical mechanisms and cannot be explained by flame temperature reduction due to added moisture since the comparison was made for the same flame temperature.
Fatigue Crack Growth Life Prediction for Surface Crack Located in Stress Concentration Part Based on the Three-Dimensional Finite Element Method
Authors: Yamashita Y. , Shinozaki M. , Ueda Y. , Sakano K.
Published: March 2004

Fatigue crack growth prediction methods using three-dimensional finite element analyses were investigated to improve the predictability of part-through surface crack growth life. First, a direct analysis method of cyclic finite element analysis was adopted. Fatigue crack growth was predicted on a step by step basis from the Paris’ law using stress intensity factor range ΔK calculated by the three-dimensional finite element method. This method takes the procedure of cyclic operation of finite element analysis modeled with crack tip elements, crack growth increment calculation and remeshing of the finite element model. Second, a method based on the influence function method for the ΔK calculation directly using three-dimensional finite element method analysis result has been developed and applied. It was found that crack growth prediction based on the step by step finite element method and the method based on the influence function method showed good correlation with the experimental results if Paris’ law coefficient C, determined by CT specimen, was appropriately used for a semi-elliptical surface crack.
Gas Turbines Design and Off-Design Performance Analysis With Emissions Evaluation
Authors: Andreini A. , Facchini B.
Published: March 2004

Many gas turbines simulation codes have been developed to estimate power plant performance both in design and off-design conditions in order to establish the adequate control criteria or the possible cycle improvements; estimation of pollutant emissions would be very important using these codes in order to determine the optimal performance satisfying legal emission restrictions. This paper present the description of a one-dimensional emission model to simulate different gas turbine combustor typologies, such as conventional diffusion flame combustors, dry-low NOx combustors (DLN) based on lean-premixed technology (LPC) or rich quench lean scheme (RQL) and the new catalytic combustors. This code is based on chemical reactor analysis, using detailed kinetics mechanisms, and it is integrated with an existing power plant simulation code (ESMS Energy System Modular Simulator) to analyze the effects of power plant operations and configurations on emissions. The main goal of this job is the study of the interaction between engine control and combustion system. This is a critical issue for all DLN combustors and, in particular, when burning low-LHV fuel. The objective of this study is to evaluate the effectiveness of different control criteria with regard to pollutant emissions and engine performances. In this paper we present several simulations of actual engines comparing the obtained results with the experimental published data.
Deterministic Micro Asperities on Bearings and Seals Using a Modified LIGA Process
Authors: Stephens L. S. , Siripuram R. , Hayden M. , McCartt B.
Published: March 2004

Deterministic micro asperities show potential for enhancement of lubrication in conformal contacts as found in many bearing and seal designs. Several manufacturing methods have been proposed for deterministic micro asperities. Of these, laser texturing has emerged as the most viable option. This paper proposes the LIGA MEMs manufacturing method as an alternative. Using LIGA, surfaces with patterned micron sized surface features of arbitrary cross section (cylindrical, hex, triangular, etc.) can be fabricated from electroplated nickel, gel-cast silicon nitride, or plastic. The resulting asperities can be positive (protuberances) or negative (recesses) and can have heights (depths) from 1–1000 microns and be patterned over surface areas up to about 150mm×150mm. In this paper, the LIGA method is used to fabricate a sample thrust bearing surface with a hexagonal array of positive asperities. The resulting asperities are 550 μm in average diameter, 165 μm in edge-to-edge spacing and have heights of 3–100 μm. Surface metrology indicates submicron accuracy of form and 13 nm Ra roughness on the asperity tops (land). Tribology testing in a nonpressurized oil bath indicates full film conditions and shows a 14–22% reduction in friction coefficient for a thrust surface covered with the micro asperities. A model confirms the experimental trends and indicates the potential to further reduce the friction coefficient by about 60% through optimization of the asperity geometry and layout.
Microturbines and Trigeneration: Optimization Strategies and Multiple Engine Configuration Effects
Authors: Campanari S. , Boncompagni L. , Macchi E.
Published: March 2004

This paper investigates energy savings and economic aspects related to the use of microturbine generators in commercial buildings either for cogeneration electricity+heat or for trigeneration (electricity, heat and cold). In all calculations, reference is made to a 25kWel-class commercial microturbine generator (MTG), tested by the authors. Various plant schemes are considered, based on one or several MTG sets. The possibility of generating heat and/or cold also by an electrically driven inverse-cycle air-to-water heat pump/chiller system is also considered. Calculations are based on the simulation code TRIGEN developed by the authors. The code provides detailed energy, economic and emission yearly balances. The plant operating mode is optimized in each time interval. The results indicate that, due to large load variations, (i) the optimum turbine nominal output is in the range of about 70% of the electric peak demand, (ii) energy savings are marginal, (iii) advantages related to splitting the overall capacity on more than one unit are marginal, and (iv) the addition of an absorption machine improves the plant economics.
Effect of Optimization Criteria on Direct-Injection Homegeneous Charge Compression Ignition Gasoline Engine Performance and Emissions Using Fully Automated Experiments and Microgenetic Algorithms
Authors: Canakci M. , Reitz R. D.
Published: March 2004

Homogeneous charge compression ignition (HCCI) is a new low-emission engine concept. Combustion under homogeneous, low equivalence ratio conditions results in modest temperature combustion products, containing very low concentrations of NOx and PM as well as providing high thermal efficiency. However, this combustion mode can produce higher HC and CO emissions than those of conventional engines. Control of the start of combustion timing is difficult with pre-mixed charge HCCI. Accordingly, in the present study charge preparation and combustion phasing control is achieved with direct injection. An electronically controlled Caterpillar single-cylinder oil test engine (SCOTE), originally designed for heavy-duty diesel applications, was converted to a direct-injection gasoline engine. The engine features an electronically controlled low-pressure direct injection-gasoline (DI-G) injector with a 60 deg spray angle that is capable of multiple injections. The use of double injection was explored for emission control, and the engine was optimized using fully automated experiments and a microgenetic algorithm optimization code. The variables changed during the optimization include the intake air temperature, start of injection timing, and the split injection parameters (percent mass of fuel in each injection, dwell between the pulses) using three different objective (merit) functions. The engine performance and emissions were determined at 700 rev/min with a constant fuel flow rate at 10 MPa fuel injection pressure. The results show the choice of merit or objective function (optimization goal) determines the engine performance, and that significant emission reductions can be achieved with optimal injection strategies. Merit function formulations are presented that minimized PM, HC, and NOx emissions, respectively.
Characteristic and Computational Fluid Dynamics Modeling of High-Pressure Gas Jet Injection
Authors: Li Y. , Kirkpatrick A. , Mitchell C. , Willson B.
Published: March 2004

The topic of this paper is the computational modeling of the gas injection process in a large-bore natural gas fueled engine. At high injection pressures, the overall gas injection and mixing process includes compressible flow features such as rarefaction waves and shock formation. The injection geometries examined in the paper include both a two-dimensional slot and an axisymmetric nozzle. The computations examine the effect of the supply pressure/cylinder stagnation pressure ratio, with ratios ranging from 3 to 80, on the velocity and pressure profiles in the near field region. Computational fluid dynamics modeling was compared with results obtained from a two-dimensional analytical method of characteristics solution and experimental results. The comparison process evaluated factors such as pressure and Mach number profiles, jet boundary shape, and shock location.
Fracture Toughness Testing on Bars Under Opposite Cylinder Loading
Authors: Fett T. , Munz D. , Thun G.
Published: March 2004

Bars loaded by opposite concentrated forces via rollers are appropriate test specimens for the determination of the fracture toughness, KIc, and the crack resistance curve (R-curve) of ceramic materials. In this paper stress solutions for the proposed test specimens are provided, as well as the stress intensity factor and the T-stress solutions. As practical applications, R-curves are determined for a soft PZT ceramic and several alumina ceramics.
The Recuperative Auto Thermal Reforming and Recuperative Reforming Gas Turbine Power Cycles With CO2 Removal—Part II: The Recuperative Reforming Cycle
Authors: Fiaschi D. , Lombardi L. , Tapinassi L.
Published: March 2004

The relatively innovative gas turbine based power cycles R-ATR and R-REF (recuperative–auto thermal reforming GT cycle and recuperative–reforming GT cycle) here proposed, are mainly aimed to allow the upstream CO2 removal by the natural gas fuel reforming. The second part of the paper is dedicated to the R-REF cycle: the power unit is a gas turbine (GT), fuelled with reformed and CO2 cleaned gas, obtained by the addition of several sections to the simple GT cycle, mainly: • reformer section (REF), where the reforming reactions of methane fuel with steam are accomplished: the necessary heat is supplied partially by the exhausts cooling and, partially, with a post-combustion, • water gas shift reactor (WGSR), where the reformed fuel is, shifted into CO2 and H2 with the addition of water, and • CO2 removal unit for the CO2 capture from the reformed and shifted fuel. No water condensing section is adopted for the R-REF configuration. Between the main components, several heat recovery units are applied, together with GT cycle recuperator, compressor intercooler, and steam injection into the combustion chamber. The CO2 removal potential is close to 90% with chemical absorption by an accurate choice of amine solution blend: the heat demand for amine regeneration is completely self-sustained by the power cycle. The possibility of applying steam blade cooling (the steam is externally added) has been investigated: in these conditions, the R-REF has shown efficiency levels close to 43–44%. High values of specific work have been observed as well (around 450–500 kJ/kg). The efficiency is slightly lower than that found for the R-ATR solution, and 2–3% lower than CRGTs with CO2 removal and steam bottoming cycle, not internally recuperated. If compared with these, the R-REF offers higher simplicity due to absence of the steam cycle, and can be regarded as an improvement to the simple GT. In this way, at least 5–6 points efficiency can be gained, together with high levels of CO2 removal. The effects of the reformed fuel gas composition, temperature, and pressure on the amine absorption system for the CO2 removal have been investigated, showing the beneficial effects of increasing pressure (i.e., pressure ratio) on the specific heat demand.
Hydraulic Lifting of Manganese Nodules Through a Riser
Authors: Xia J. X. , Ni J. R. , Mendoza C.
Published: March 2004

The calculation of the hydraulic gradient due to the upward flow of a large size particles—water mixture in a vertical pipe is a central problem in the design of systems for deep-sea mining of manganese nodules. Here, the problem is investigated experimentally and with a new calculation method. An experimental apparatus that mimics the deep-sea mining system was built to measure the hydraulic gradient due to the mixture upward flow, the settling velocity of a single manganese nodule, and to explore the relationship between the concentration of fluidized manganese nodules and the solid slip velocity. Experimental relations are found. Also, a formula to compute the total hydraulic gradient of the mixture flow under different flow and solid-loading conditions is developed; the formula accounts for the hydraulic gradients produced by the liquid phase, the solid phase, and the inter-particle collisions. The predictions obtained with the derived equation are compared with experimental data readily available and with the newly acquired laboratory data; these predictions agree very well with the empirical data and demonstrate the value of the model as a design tool.
The Diffraction of Multidirectional Random Waves by Rectangular Submarine Pits
Authors: Lee Hong Sik , Williams A. Neil
Published: March 2004

The diffraction of multidirectional random surface waves with one or more rectangular submarine pits is investigated theoretically. The incident wave conditions are specified using a discrete form of the Mitsuyasu directional spectrum. The numerical model involves the superposition of regular-wave diffraction solutions based on linearized shallow water wave theory obtained by a two-dimensional boundary integral approach for water of uniform depth. Numerical results are presented for multi-directional random waves that illustrate the effect of the various wave and pit parameters on the diffraction characteristics of typical single and multiple pit systems.
Wave Crest Sensor Intercomparison Study: An Overview of WACSIS
Authors: Forristall George Z. , Barstow Stephen F. , Krogstad Harald E. , Prevosto Marc , Taylor Paul H. , Tromans Peter S.
Published: March 2004

The Wave Crest Sensor Intercomparison Study (WACSIS) was designed as a thorough investigation of the statistical distribution of crest heights. Measurements were made in the southern North Sea during the winter of 1997–1998 from the Meetpost Noordwijk in 18 m water depth. The platform was outfitted with several popular wave sensors, including Saab and Marex radars, an EMI laser, a Baylor wave staff and a Vlissingen step gauge. Buoys were moored nearby to obtain directional spectra. Two video cameras viewed the ocean under the wave sensors and their signals were recorded digitally. The data analysis focused on comparisons of the crest height measurements from the various sensors and comparisons of the crest height distributions derived from the sensors and from theories. Some of the sensors had greater than expected energy at high frequencies. Once the measurements were filtered at 0.64 Hz, the Baylor, EMI and Vlissingen crest height distributions matched quite closely, while those from the other sensors were a few percent higher. The Baylor and EMI crest distributions agreed very well with the statistics from second order simulations, while previous parameterizations of the crest height distribution were generally too high. We conclude that crest height distributions derived from second order simulations can be used with confidence in engineering calculations. The data were also used in investigations of crest and trough shapes and the joint height/period distribution.
Nonlinear Hydrodynamic Forces on Flexible Structures: A Combined-Force Model
Authors: Anam Iftekhar , Roesset Jose M.
Published: March 2004

A new combined-force method is suggested to approximate the second-order difference frequency forces from diffraction theory (Φ2 theory) with less computational effort. The new method is formulated by combining two limiting cases of the Φ2 theory; i.e., Newman’s approximation and the slender Φ2 theory. Numerical results show that the new method reproduces the individual nonlinear effects of the Φ2 theory better than the existing approximations. Results of this work also show the limitations of Morison’s equation as the slender-body counterpart of the diffraction theory for nonlinear problems.
Effect of Material Stress-Strain Behavior and Pipe Geometry on the Deformability of High-Grade Pipelines
Authors: Yatabe Hiroshi , Fukuda Naoki , Masuda Tomoki , Toyoda Masao
Published: March 2004

In this study, the deformability of high-grade pipelines subjected to an axial compressive deformation was experimentally and analytically discussed. Six cases of axial compression experiments with high-grade line pipe were carried out. The pipe specimens had various material properties and wall thickness. Finite-element analyses were also carried out and verified the reliability. Then, a finite-element analysis method for evaluating the deformability of the line pipe was established. By using this method, parametric studies were carried out. The effects of the strain-hardening behavior and pipe geometry on the deformability of the high-grade pipelines were examined.
Hydroelastic Instability of Low Aspect Ratio Control Surfaces
Authors: McCormick Michael E. , Caracoglia Luca
Published: March 2004

As the operational speeds of surface ships and submarines increase, so does the probability that unwanted vibrations caused by the hydroelastic instability (flutter) of the special class of hydrofoils called control surfaces. These include rudders and diving planes. By nature, these are thick symmetric hydrofoils having low aspect ratios. The 3-D tip effects become more pronounced as the aspect ratio decreases. In the present study, the added-mass and circulation terms of the 2-D flutter equations are modified to include three-dimensional effects. The modifications are performed by introducing quasi-steady coefficients to each term. The results predicted by the modified equations are found to compare well with experimental results on a towed rectangular foil having an aspect ratio of one.
Mitigation of Current-Driven, Vortex-Induced Vibrations of a Spar Platform via “SMART” Thrusters
Authors: Fischer F. Joseph , Liapis Stergios I. , Kallinderis Yannis
Published: March 2004

The possibility of using active-control technology to mitigate long-period (100–300 s) motions of spar platforms was investigated. In particular, the technical feasibility and cost-effectiveness of using a thruster-based active-control system was examined. Only vortex-induced vibrations (VIV) of a moored spar were considered for this study, although it is believed that similar active-control systems can contend equally well with low-frequency wave-drift and wind-induced motions. VIV has been selected for this demonstration due to the considerable attention it has received for spars, because of the high cost for passive VIV-mitigation means. The investigation employed numerical simulations of the hydrodynamics around the spar in two and three dimensions. In particular, for this study, Navier-Stokes methods were used to compute the strongly nonlinear interactions between the current-flow field and the spar hull. The fluid forces (hull-integrated pressures) were then used to compute the unsteady motions of the moored spar. The effect of the motion-controlling thrusters was included as an additional external force—that also varies with time. Of the several different control strategies that were examined, it was found that the most effective one is “D-Control” wherein thruster forces are proportional to spar velocities. Using such control, spar motions can be kept below 35% of the spar diameter, whereas uncontrolled responses were computed to be as high as 80% of the spar diameter. Similar uncontrolled motions have actually been observed in current-tank model tests of spars. As part of this study, discussions were held with engineers from three different thruster companies to ascertain the number and type of thrusters that would be required to achieve desirable VIV mitigation—given the computed thruster-forces, and to provide prudent reliability. Associated costs were also very much of interest. For the spar system examined here, which is representative of those introduced into or being considered for the Gulf of Mexico, it was found that the cost of the thrusters and their electric motors is around $8 million—a cost that is significantly less than that estimated for passive VIV-control devices such as helical strakes, or discontinuous hull geometries. Furthermore, the proposed thruster system could also be employed to contend with (to mitigate) other undesirable, large-amplitude, near-resonant spar motions, e.g., low-frequency drift, for which there are no alternative remedies at present.
Wave Period Distribution in Mixed Sea-States
Authors: Rodrı´guez Germa´n , Guedes Soares C. , Pacheco Mercedes
Published: March 2004

The statistical distribution of zero up-crossing wave periods in Gaussian mixed sea-states is examined by using numerically simulated data. Nine different kinds of bimodal spectra are used to analyze the effects of the relative energy ratio and the peak frequency separation between the low- and high-frequency wave fields on the wave period distribution. Observed results are compared with predictions given by probabilistic models suitable for practical applications. Numerical results reveal a different behavior for low- and high-period bands. Furthermore, comparisons of the empirical data with some probabilistic models often used in practice shows that none of these models is able to characterize adequately all the cases of combined sea-states examined. In fact, they might be used only in the case of wind-sea dominated sea-states with small separation between spectral frequency peaks. However, a model recently proposed by Myrhaug and Slaattelid [1] represents adequately the observed distributions of wave period.
Finite-Element Modelling of Multi-Planar Offshore Tubular Joints
Authors: Lee Marcus M. K. , Dexter Ellen M.
Published: March 2004

This paper describes the calibration and validation of a finite-element modelling procedure that was used to conduct an extensive parametric study on the strength of multi-planar tubular joints. Various factors that influenced results were investigated. The modelling procedure was calibrated using the International Organization for Standards (ISO) design equations and the underlying test database for compression and tension loaded simple T and Y joints, and was further validated with balanced loaded K joints. Overall, the proposed procedure has been shown to be adequate in predicting the strength of the basic joint types, thereby giving confidence in its use for more complex joints.
Discovery of a Large Dust Disk Around the Nearby Star AU Microscopii
Authors: Kalas Paul , Liu Michael C. , Matthews Brenda C.
Published: March 2004

We present the discovery of a circumstellar dust disk surrounding AU Microscopii (AU Mic, GJ 803, HD 197481). This young M star at 10 parsec has the same age and origin as β Pictoris, another nearby star surrounded by a dust disk. The AU Mic disk is detected between 50 astronomical units (AU) and 210 AU radius, a region where dust lifetimes exceed the present stellar age. Thus, AU Mic is the nearest star where we directly observe the solid material required for planet formation. Because 85% of stars are M-type, the AU Mic disk provides new clues on how the majority of planetary systems might form and evolve.
Chimeric Nature of Two Plasmids of
Hafnia alvei
Encoding the Bacteriocins Alveicins A and B
Authors: Wertz John E. , Riley Margaret A.
Published: March 2004

The complete nucleotide sequences of two bacteriocin-encoding plasmids isolated from
Hafnia alvei
(pAlvA and pAlvB) were determined. Both plasmids resemble ColE1-type replicons and carry mobilization genes, as well as colicin-like bacteriocin operons. These bacteriocins appear to be chimeras consisting of translocation domains from Tol-dependent colicins, unique binding domains, and killing and immunity domains similar to those of the pore-forming colicin Ia. Just as is found for colicin Ia, these
H. alvei
bacteriocins (alveicins) lack lysis genes. The alveicins are unusually small at 408 and 358 amino acids for alveicin A and B, respectively, which would make alveicin B the smallest pore-forming bacteriocin yet discovered. The pattern of nucleotide substitution in the alveicins suggests that the dominant forces in the evolution of their killing domains and immunity genes are neutral mutation and random genetic drift rather than diversifying selection, which has been implicated in the evolution of other colicins. Five of six bacteriocinogenic isolates of
H. alvei
were found to carry plasmids identical to pAlvA. Comparisons of the levels of nucleotide divergence in five housekeeping genes to the levels of divergence in their respective plasmids led us to conclude that pAlvA is transferring laterally through the
H. alvei
population relatively rapidly.
Regulation ofEscherichia coliHemolysin E Expression by H-NS andSalmonellaSlyA
Authors: Wyborn Neil R. , Stapleton Melanie R. , Norte Valia A. , Roberts Ruth E. , Grafton Jamie , Green Jeffrey
Published: March 2004

TheEscherichia coli hlyEgene (also known asclyAorsheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulatehlyEexpression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies thehlyEpromoter more frequently than CRP, providing a mechanism for the moderate upregulation ofhlyEexpression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of thehlyEpromoter (PhlyE), one upstream of the −35 element and one downstream of the −10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3′ end of the extended upstream footprint, and H-NS II, located at the 5′ end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyEsuggests a mechanism to explain how SlyA overproduction enhanceshlyEexpression by antagonizing the negative effects of H-NS.
Phosphatidylethanolamine Is Not Essential for Growth of
Sinorhizobium meliloti
on Complex Culture Media
Authors: Sohlenkamp Christian , de Rudder Karel E. E. , Geiger Otto
Published: March 2004

In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE),
Sinorhizobium meliloti
also possesses phosphatidylcholine (PC) as a major membrane lipid. The biosynthesis of PC in
S. meliloti
can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly. Therefore, for
S. meliloti
, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis. In this study, we investigated the first step of PE biosynthesis in
S. meliloti
catalyzed by phosphatidylserine synthase (PssA). A sinorhizobial mutant lacking PE was complemented with an
S. meliloti
gene bank, and the complementing DNA was sequenced. The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases. Inactivation of the sinorhizobial
pssA
gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type. A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions. On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation. Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype.
DNA Amplification and Rearrangements in Archival
Salmonella enterica
Serovar Typhimurium LT2 Cultures
Authors: Porwollik Steffen , Wong Rita Mei-Yee , Helm R. Allen , Edwards Kelly K. , Calcutt Michael , Eisenstark Abraham , McClelland Michael
Published: March 2004

Variations in genome size and gene order were observed in archival
Salmonella enterica
serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.
Mutational Analysis of the Signal-Sensing Domain of ResE Histidine Kinase fromBacillus subtilis
Authors: Baruah Avanti , Lindsey Brett , Zhu Yi , Nakano Michiko M.
Published: March 2004

TheBacillus subtilisResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO). The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription. ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain. The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop. It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain. In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain. The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes. This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception. The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain. This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism inB. subtilis.
Regulation of Expression of the Divergent
ulaG
and
ulaABCDEF
Operons Involved in
l
-Ascorbate Dissimilation in
Escherichia coli
Authors: Campos Evangelina , Baldoma Laura , Aguilar Juan , Badia Josefa
Published: March 2004

The
ula
regulon, responsible for the utilization of
l
-ascorbate in
Escherichia coli
, is formed by two divergently transcribed operons,
ulaG
and
ulaABCDEF
. The regulon is negatively regulated by a repressor of the DeoR family which is encoded by the constitutive gene
ulaR
located downstream of
ulaG
. Full repression of the
ula
regulon requires simultaneous interaction of the repressor with both divergent promoters and seems to be dependent on repressor-mediated DNA loop formation, which is helped by the action of integration host factor. Two operator sites have been identified in each promoter. Lack of either of the two sets of operators partially relieved the repression of the other operon; thus, each promoter is dependent on the UlaR operator sites of the other promoter to enhance repression. Electrophoretic mobility shift assays with purified UlaR protein and promoter deletion analyses revealed a conserved sequence, present in each of the four operators, acting as a UlaR binding site. Glucose represses the
ula
regulon via at least two mechanisms, one dependent on cyclic AMP (cAMP)-cAMP receptor protein (CRP) and the other (possibly inducer exclusion) independent of it. Glucose effects mediated by other global regulators cannot be ruled out with the present information. Changes in cAMP-CRP levels affected only the expression of the
ulaABCDEF
operon.
Sustained Photoevolution of Molecular Hydrogen in a Mutant of
Synechocystis
sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex
Authors: Cournac Laurent , Guedeney Geneviève , Peltier Gilles , Vignais Paulette M.
Published: March 2004

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of
Synechocystis
sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H
2
, CO
2
, and O
2
) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H
2
:NAD(P) oxidoreductase], encoded by the
hoxEFUYH
genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D
2
. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O
2
, and could be quickly reactivated by NADH or NADPH (+H
2
). H
2
was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H
2
evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H
2
output was observed, followed by rapid H
2
uptake and a concomitant decrease in CO
2
concentration in the cyanobacterial cell suspension. Uptake of both H
2
and CO
2
was linked to photosynthetic electron transport in the thylakoids. In the
ndhB
mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O
2
in the light, H
2
uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H
2
production. A sustained rate of photoevolution of H
2
corresponding to 6 μmol of H
2
mg of chlorophyll
−1
h
−1
or 2 ml of H
2
liter
−1
h
−1
was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O
2
scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone), it was shown that two pathways of electron supply for H
2
production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.
Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of
Geobacillus stearothermophilus
PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology
Authors: Mader Christoph , Huber Carina , Moll Dieter , Sleytr Uwe B. , Sára Margit
Published: March 2004

The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of
Geobacillus stearothermophilus
PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains
N
-acetylglucosamine,
N
-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall
K

d

of 7.7 × 10
−7
M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (
K

d

= 2.6 × 10
−5
M), medium (
K

d

= 6.1 × 10
−8
M), and high (
K

d

= 6.7 × 10
−11
M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.
Accessory Gene Regulator Control of Staphyloccoccal Enterotoxin D Gene Expression
Authors: Tseng Ching Wen , Zhang Shuping , Stewart George C.
Published: March 2004

The quorum-sensing system of
Staphylococcus aureus
, the accessory gene regulator (Agr) system, is responsible for increased transcription of certain exoprotein genes and decreased transcription of certain cell wall-associated proteins during the postexponential phase of growth. This regulation is important for virulence, as evidenced by a reduction in virulence associated with a loss of the Agr system. The enterotoxin D (
sed
) determinant is upregulated by the Agr system. To define the Agr-regulated
cis
element(s) within the
sed
promoter region, we utilized promoters not regulated by Agr to create hybrid promoters. Hybrid promoters were created by using
sed
sequences combined with the enterotoxin A (
sea
) promoter or the
S. aureus lac
operon promoter sequences. The results obtained indicated that the Agr control element of the
sed
promoter resides within the −35 promoter element and at the Pribnow box to the +1 site of the promoter. At these positions of the
sed
promoter, a directly repeated 6-bp sequence was found. This repeat is important for overall promoter activity, and maximal regulation of the promoter activity requires both repeat elements. Furthermore, Agr control of
sed
promoter activity was found to be dependent upon the presence of a functional Rot protein. Therefore, the postexponential increase in
sed
transcription results from the Agr-mediated reduction in Rot activity rather than as a direct effect of the Agr system.
Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues
Authors: Marshall Samantha J. , Krause Doreen , Blencowe Dayle K. , White Graham F.
Published: March 2004

Glycerol trinitrate reductase (NerA) from
Agrobacterium radiobacter
, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from
Escherichia coli
. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10
−7
M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same
k
cat
as that of wild-type enzyme but with altered

K
m

values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of

K
m

GTN
to

K
m

NAD(P)H
, with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.
Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in
Methanothermobacter thermautotrophicus
Authors: Barkley Sam J. , Cornish Rita M. , Poulter C. Dale
Published: March 2004

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in
Streptomyces
sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon
Methanothermobacter thermautotrophicus
encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in
Salmonella enterica
serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (
idi
) and 1-deoxy-
d
-xylulose 5-phosphate (
dxs
). The
dxs
gene was interrupted with a synthetic operon containing the
Saccharomyces cerevisiae
genes
erg8
,
erg12
, and
erg19
allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His
6
-tagged
M. thermautotrophicus
type II IPP isomerase was produced in
Escherichia coli
and purified by Ni
2+
chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg
2+
are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from
M. thermautotrophicus
are as follows:

K
m

, 64 μM; specific activity, 0.476 μmol mg
−1
min
−1
; and
k
cat
, 1.6 s
−1
.
The pKO2 Linear Plasmid Prophage ofKlebsiella oxytoca
Authors: Casjens Sherwood R. , Gilcrease Eddie B. , Huang Wai Mun , Bunny Kim L. , Pedulla Marisa L. , Ford Michael E. , Houtz Jennifer M. , Hatfull Graham F. , Hendrix Roger W.
Published: March 2004

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bpKlebsiella oxytocalinear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage φKO2. An analysis of the 64 predicted φKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The φKO2 virion is unusual in that its phage λ-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The φKO2 genome also carries putative homologues of bacterialdinIandumuDgenes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.