bluuebunny/crossref_metadata_2025 huggingface dataset:
Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling
Authors: Houtman Jon C.D. , Brown Patrick H. , Bowden Brent , Yamaguchi Hiroshi , Appella Ettore , Samelson Lawrence E. , Schuck Peter
Published: December 2006

Multisite interactions and the formation of ternary or higher‐order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T‐cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T‐cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.
The True Linearization Coefficients for Nonlinear Systems Under Parametric White Noise Excitations
Authors: Falsone Giovanni
Published: December 2006

In this paper some properties of the stochastic linearization method applied to nonlinear systems excited by parametric Gaussian white noises are discussed. In particular, it is shown that the linearized quantities, obtained by the author in another paper by linearizing the coefficients of the Ito differential rule related to the original system, show the same properties found by Kozin with reference to nonlinear system excited by external white noises. The first property is that these coefficients are the true linearized quantities, in the sense that their exact values are able to give the first two statistical moments of the true response. The second property is that, in the stationary case and in the field of the parameter estimation theory, they represent the maximum likelihood estimates of the linear model quantities fitting the original nonlinear response.
Spectral Characteristics of the Near-Wall Turbulence in an Unsteady Channel Flow
Authors: Tardu Sedat F.
Published: December 2006

The modulation characteristics of the turbulent wall shear stress and longitudinal intensities in the inner layer are experimentally investigated in an unsteady channel flow wherein the centerline velocity varies in time in a sinusoidal manner. The fluctuating wall shear stress and velocity signals are temporally filtered and subsequently phase averaged. It is shown that the outer structures corresponding to the low spectrum range have a constant time lag with respect to the centerline velocity modulation. The inner active structures, in particular those with a frequency band containing the mean ejection frequency of the corresponding steady flow dominate the dynamics of the near-wall unsteady turbulence. The structures respond to the imposed shear oscillations in a complex way, depending both on their characteristic scales and the thickness of the oscillating shear zone in which they are embedded.
A Greenwood-Williamson Model of Small-Scale Friction
Authors: Jones Reese E.
Published: December 2006

A Greenwood and Williamson based model for interfacial friction is presented that incorporates the presliding transition phenomenon that can significantly affect small devices. This work builds on previous similar models by developing: an analytical estimate of the transition length in terms of material and surface parameters, a general recursion formula for the case of slip in one direction with multiple reversals and constant normal loading, and a numerical method for the general three-dimensional loading case. In addition, the proposed model is developed within a plasticity-like framework and is shown to have qualitative similarities with published experimental observations. A number of model problems illustrate the response of the proposed model to various loading conditions.
Electromagnetoelastic Dynamic Response of Transversely Isotropic Piezoelectric Hollow Spheres in a Uniform Magnetic Field
Authors: Dai H. L. , Fu Y. M. , Liu T. X.
Published: December 2006

An analytical method is presented to solve the problem of electromagnetoelastic dynamic response of transversely isotropic piezoelectric hollow spheres in a uniform magnetic field, subjected to arbitrary mechanical load and electric excitation. Exact expressions for the dynamic responses of stresses, perturbation of magnetic field vector, electric displacement, and electric potential in piezoelectric hollow spheres are obtained by means of Hankel transform, Laplace transform and their inverse transforms. An interpolation method is applied to solve the Volterra integral equation of the second kind involved in the exact expression, which is caused by interaction between electric-elastic field and electric-magnetic field. Thus, an analytical solution for the problem of dynamic response of a transversely isotropic piezoelectric hollow sphere in a uniform magnetic field is obtained. Finally, some numerical examples are carried out, and may be used as a reference to solve other dynamic coupled problems of electromagneto-elasticity.
On the Admissibility of Given Acceleration-Dependent Forces in Mechanics
Authors: Zhechev Michael M.
Published: December 2006

In his book “A Treatise on Analytical Dynamics,” Pars asserted that acceleration-dependent forces are inconsistent with one of the fundamental principles of mechanics, namely, with the superposition principle, thus spreading among mechanical scientists the idea that such forces are not admissible in mechanics. This article demonstrates that given forces that depend on acceleration or higher derivatives are admissible in mechanics and shows that this assertion in Pars’s book is fallacious and the only condition for the applicability of such forces is the equation of motion possessing a unique solution.
Computer Simulation of Rapid Granular Flow Through an Orifice
Authors: Ahn Hojin
Published: December 2006

Rapid granular flow through an orifice (nozzle-shaped flow restrictor) located at the bottom of a vertical tube has been studied using three-dimensional direct computer simulation with the purpose of investigating (1) characteristics of rapid granular flows through the flow restrictor, (2) the choking condition of rapid flow at the orifice and thus conditions at which the maximum discharge rate takes place for the given orifice, and (3) a functional relationship between the discharge rate and flow quantities such as granular temperature and solid fraction. In the present simulation, where the frictional hard-sphere collision operator was employed, it was possible to obtain both rapid and slow (choked) flows through the orifice by controlling the number of particles in the system. The results show that the profile of granular temperature in the vicinity of the orifice plays an important role in determining the choking condition at the orifice. Flow appears to be choked when an adverse granular conduction occurs locally at the orifice in the direction opposite to the mean flow. On the other hand, flow is not choked when the fluctuation energy is conducted in the mean flow direction near the orifice. When flow is not choked, the discharge rate through the orifice increases with increasing solid fraction or normal stress. Once the flow becomes choked, however, the discharge rate decreases as the solid fraction or normal stress increases. Also for inelastic, rough particles, the discharge rate is found to be proportional to the granular temperature to the power of 1.5 and inversely proportional to the gravitational acceleration and the tube length.
Plane Analysis of Finite Multilayered Media With Multiple Aligned Cracks—Part I: Theory
Authors: Chen Linfeng , Pindera Marek-Jerzy
Published: December 2006

Elasticity solutions are developed for finite multilayered domains weakened by aligned cracks that are in a state of generalized plane deformation under two types of end constraints. Multilayered domains consist of an arbitrary number of finite-length and finite-height isotropic, orthotropic or monoclinic layers typical of differently oriented, unidirectionally reinforced laminas arranged in any sequence in the plane in which the analysis is conducted. The solution methodology admits any number of arbitrarily distributed interacting or noninteracting cracks parallel to the horizontal bounding surfaces at specified elevations or interfaces. Based on half-range Fourier series and the local/global stiffness matrix approach, the mixed boundary-value problem is reduced to a system of coupled singular integral equations of the Cauchy type with kernels formulated in terms of the unknown displacement discontinuities. Solutions to these integral equations are obtained by representing the unknown interfacial displacement discontinuities in terms of Jacobi or Chebyshev polynomials with unknown coefficients. The application of orthogonality properties of these polynomials produces a system of algebraic equations that determines the unknown coefficients. Stress intensity factors and energy release rates are derived from dominant parts of the singular integral equations. In Part I of this paper we outline the analytical development of this technique. In Part II we verify this solution and present new fundamental results relevant to the existing and emerging technologies.
Optimization Study for a Parallel Plate Impingement Heat Sink
Authors: Shah Amit , Sammakia Bahgat G. , Srihari K. , Ramakrishna K.
Published: December 2006

A previous study by the authors on fin-shape optimization of a plate fin heat sink has concluded that a depopulated central zone, just under the center of the fan, provides a better thermal performance compared to the heat sink geometries with fin material under the fan. This study extends the previous work by investigating the effect of removal of fin material from the end fins, the total number of fins, and the reduction in the size of the hub fan. From this study, it is concluded that the removal of fin material from the end fins results in a better thermal and hydraulic performance of the heat sink. The reduction in the size of the hub causes a more uniform distribution of air inside the heat sink. The increase in the number of fins indicates a slightly better thermal performance, accompanied by a considerably increased pressure drop. Finally, a new optimal heat sink design has been reported by employing the actual fan operating characteristics.
Prognostics Assessment of Aluminum Support Structure on a Printed Circuit Board
Authors: Mathew Sony , Das Diganta , Osterman Michael , Pecht Michael , Ferebee Robin
Published: December 2006

This paper presents an analysis of an unexpected failure during vibration and shock life test of an electronic circuit board that has been in use for more than 15 years. During testing, an aluminum bracket used to mount a transistor and provide a path for heat transfer was damaged. Prognostic methods were employed to determine whether the bracket failure could have been predicted prior to the life test. Details of the analytical calculations and modeling results are described in this paper. Results show that the failure could have been predicted before actual testing.
Design of Air and Liquid Cooling Systems for Electronic Components Using Concurrent Simulation and Experiment
Authors: Icoz Tunc , Verma Nitin , Jaluria Yogesh
Published: December 2006

The design of cooling systems for electronic equipment is getting more involved and challenging due to increase in demand for faster and more reliable electronic systems. Therefore, robust and more efficient design and optimization methodologies are required. Conventional approaches are based on sequential use of numerical simulation and experiment. Thus, they fail to use certain advantages of using both tools concurrently. The present study is aimed at combining simulation and experiment in a concurrent manner such that outputs of each approach drive the other to achieve better engineering design in a more efficient way. In this study, a relatively simple problem, involving heat transfer from multiple heat sources simulating electronic components and located in a horizontal channel, was investigated. Two experimental setups were fabricated for air and liquid cooling experiments to study the effects of different coolants. De-ionized water was used as the liquid coolant in one case and air in the other. The effects of separation distance and flow conditions on the heat transfer and on the fluid flow characteristics were investigated in detail for both coolants. Cooling capabilities of different cooling arrangements were compared and the results from simulations and experiments were combined to create response surfaces and to find the optimal values of the design parameters.
Thermal Management of Electronics in Telecommunications Products: Designing for the Network Equipment Building System (NEBS) Standards
Authors: Rhee Jinny , Hernandez Sergio I.
Published: December 2006

The effects of altitude, increased ambient temperature, and increased relative humidity on the board level forced convection typical of telecommunications products were systematically examined through numerical simulation and analytical techniques. Altitude was found to have the most significant impact on component temperature rise above inlet air temperature. Depending on the proportion of upstream-heating to self-heating for a given component, the component temperature rise above inlet air temperature was found to increase by 40% to 88% at 5000m when compared to the base line case at sea level. Inlet air temperature was found to translate linearly to component temperature increase. The second-order effects due to property changes with temperature were found to be less than 3% on the component temperature rise. The relative humidity was not found to significantly impact the component temperature rise at an inlet temperature of 25°C. However, the property changes at a 55°C inlet were more significant, and require further study. A temperature multiplier concept is introduced to aid practicing engineers in extrapolating data at standard conditions to the operating extremes.
Optimization of Data Center Room Layout to Minimize Rack Inlet Air Temperature
Authors: Bhopte Siddharth , Agonafer Dereje , Schmidt Roger , Sammakia Bahgat
Published: December 2006

In a typical raised floor data center with alternating hot and cold aisles, air enters the front of each rack over the entire height of the rack. Since the heat loads of data processing equipment continue to increase at a rapid rate, it is a challenge to maintain the temperature of all the racks within the stated requirement. A facility manager has discretion in deciding the data center room layout, but a wrong decision will eventually lead to equipment failure. There are many complex decisions to be made early in the design as the data center evolves. Challenges occur such as optimizing the raised floor plenum, floor tile placement, minimizing the data center local hot spots, etc. These adjustments in configuration affect rack inlet air temperatures, which is one of the important keys to effective thermal management. In this paper, a raised floor data center with 12kW racks is considered. There are four rows of racks with alternating hot and cold aisle arrangement. Each row has six racks installed. Two air-conditioning units supply chilled air to the data center through the pressurized plenum. Effect of plenum depth, floor tile placement, and ceiling height on the rack inlet air temperature is discussed. Plots will be presented over the defined range. A multivariable approach to optimize data center room layout to minimize the rack inlet air temperature is proposed. Significant improvement over the initial model is shown by using a multivariable design optimization approach.
Some Aspects on Hot Forging Features of P/M Sintered High-Strength Titanium Carbide Composite Steel Preforms Under Different Stress State Conditions
Authors: Narayanasamy R. , Senthilkumar V. , Pandey K. S.
Published: December 2006

Experiments were carried out to evaluate the hot forging features in the high-strength sintered Powder Metallurgy TiC composite steel performs under different stress states, namely, plane and triaxial stress. Cylindrical compacts with aspect ratios 0.45, 0.75, and 1.25 were prepared, sintered and forged at the temperatures of 1120°C+10°C. The investigation suggests that the experimentally determined relative density has a straight line relationship with the new geometrical shape factor (ρ0∕ρth)(h0∕hf)(3D02∕(2Db2+Dc2)). The measured barrel radius of curvature is found to have a circular arc because the above relation is a straight line one. Relationship is established between the measured barrel radius and the stress ratio parameters namely (σθ∕σz), (σz∕σm) and (σeff∕σz) under plane stress and triaxial stress state conditions. An attempt is also made to establish a relationship between the various stress ratio parameters namely (σθ∕σz), (σz∕σm), and (σeff∕σz) under plane stress and triaxial stress state conditions and the relative density.
Methodology for Determining the Variance of the Taylor Factor: Application in Fe-3%Si
Authors: Przybyla Craig P. , Adams Brent L. , Miles Michael P.
Published: December 2006

A method is proposed to determine the variance of an arbitrary material property based on the statistics of the texture of polycrystalline materials for a specified volume. This method is applied to determine the variance of the Taylor factor (i.e., measure of plastic deformation in crystal plasticity) and is compared to a random sampling method. The results from the random sampling method correlated well with the statistical variance relationship when the magnitude of the variance was greater than that of the numerical errors observed in the statistical calculation. An empirical relation was also shown to model the results, and the constants for this relationship were determined for pseudo-three-dimensional Fe-3%Si. Implementation of the statistical variance relationship in true three-dimensional microstructures is not limited by material opacity, since it depends only on the two-point pair correlation functions. The connection between the variance of the R-value and variance of the Taylor factor is considered. Although only a weak connection was found, it was observed that relatively small variations in the Taylor factor yield large variances in the R-value.
Amodiaquine and Artemether-Lumefantrine Select Distinct Alleles of the
Plasmodium falciparum mdr1
Gene in Tanzanian Children Treated for Uncomplicated Malaria
Authors: Humphreys G. S. , Merinopoulos I. , Ahmed J. , Whitty C. J. M. , Mutabingwa T. K. , Sutherland C. J. , Hallett R. L.
Published: December 2006

The artemisinin-based combination therapies artemether-lumefantrine (AL) and amodiaquine (AQ) plus artesunate have been adopted for treatment of
Plasmodium falciparum
malaria in many African countries. Molecular markers of parasite resistance suitable for surveillance have not been established for any of the component drugs in either of these combinations. We assessed
P. falciparum mdr1
(Pf
mdr1
) alleles present in 300 Tanzanian children presenting with uncomplicated falciparum malaria, who were enrolled in a clinical trial of antimalarial therapy. Pf
mdr1
genotype analysis was also performed with isolates from 182 children who failed AQ monotherapy and 54 children who failed AL treatment. Pf
mdr1
alleles 86Y, 184Y, and 1246Y were more common among treatment failures in the AQ group than among pretreatment infections. The converse was found in the AL-treated group. Children presenting with the 86Y/184Y/1246Y Pf
mdr1
haplotype and treated with AQ were significantly more likely to retain this haplotype if they were parasite positive during posttreatment follow-up than were children treated with AL (odds ratio, 33.25; 95% confidence interval, 4.17 to 1441;
P
, <0.001). We conclude that AL and AQ exert opposite within-host selective effects on the Pf
mdr1
gene of
P. falciparum
.
In Vitro Activities of Tigecycline and Eight Other Antimicrobials against Different
Nocardia
Species Identified by Molecular Methods
Authors: Cercenado Emilia , Marín Mercedes , Sánchez-Martínez Mónica , Cuevas Oscar , Martínez-Alarcón José , Bouza Emilio
Published: December 2006

The in vitro activities of tigecycline and other antimicrobials against 51 isolates of
Nocardia
spp. were evaluated. MIC
90
s and MIC ranges were as follows: tigecycline, 4 and ≤0.06 to 8 mg/liter, respectively; minocycline, 2 and ≤0.06 to 2 mg/liter, respectively; linezolid, 1 and ≤0.06 to 2 mg/liter, respectively; moxifloxacin, 2 and ≤0.06 to >64 mg/liter, respectively; ertapenem, 32 and ≤0.06->64 mg/liter, respectively; imipenem, 2 and ≤0.06 to >64 mg/liter, respectively; meropenem, 8 and ≤0.06 to >64 mg/liter, respectively; amikacin, 1 and ≤0.06 to 32 mg/liter, respectively; and trimethoprim-sulfamethoxazole, 1/19 and ≤0.5/9.5 to >2/38 mg/liter, respectively.
Similar Trends of Pyrimethamine Resistance-Associated Mutations in
Plasmodium vivax
and
P. falciparum
Authors: Alam Mohammad Tauqeer , Bora Hema , Bharti Praveen K. , Saifi Muheet A. , Das Manoj K. , Dev Vas , Kumar Ashwani , Singh Neeru , Dash Aditya P. , Das Brahmananda , Wajihullah  , Sharma Yagya D.
Published: December 2006

The antifolate drugs sulfadoxine and pyrimethamine are commonly used to treat
Plasmodium falciparum
malaria. However, they can also affect the
Plasmodium vivax
parasite if it coexists with
P. falciparum
, as both species have common drug targets. Resistance to the antifolate drugs arises due to point mutations in the target enzymes of the respective parasite. To assess the cross-species impact of antifolate drug treatment, we describe here the dihydrofolate reductase (DHFR) mutations among field isolates of
P. vivax
and
P. falciparum
. The overall DHFR mutation rate for
P. vivax
was lower than that for
P. falciparum
. However, both species of
Plasmodium
followed similar trends of DHFR mutations. Similar to
P. falciparum
, the DHFR mutation rate of
P. vivax
also varied from region to region. It was lower in
P. vivax
-dominant regions but higher in the
P. falciparum
-dominated areas and highest where antifolates are used as the first line of antimalarial treatment. In conclusion, the antifolate treatment of falciparum malaria is proportionately affecting the DHFR mutations of
P. vivax
, suggesting that the drug should be used with caution to minimize the development of cross-species resistance in the field.
Pretreatment with Empty Liposomes Attenuates the Immunopathology of Invasive Pulmonary Aspergillosis in Corticosteroid-Immunosuppressed Mice
Authors: Lewis Russell E. , Chamilos Georgios , Prince Randall A. , Kontoyiannis Dimitrios P.
Published: December 2006

In a nonneutropenic murine model of invasive pulmonary aspergillosis, pretreatment with empty liposomes (E-lipo) was nearly as effective as 10 mg/kg of body weight liposomal amphotericin B and superior to 1 mg/kg amphotericin B deoxycholate. The beneficial immunomodulatory properties of E-lipo appear to compensate for their lack of direct antifungal activity.
Salivaricin A2 and the Novel Lantibiotic Salivaricin B Are Encoded at Adjacent Loci on a 190-Kilobase Transmissible Megaplasmid in the Oral Probiotic Strain
Streptococcus salivarius
K12
Authors: Hyink Otto , Wescombe Philip A. , Upton Mathew , Ragland Nancy , Burton Jeremy P. , Tagg John R.
Published: December 2006

The commercial probiotic
Streptococcus salivarius
strain K12 is the prototype of those
S. salivarius
strains that are the most strongly inhibitory in a standardized test of streptococcal bacteriocin production and has been shown to produce the 2,368-Da salivaricin A2 (SalA2) and the 2,740-Da salivaricin B (SboB) lantibiotics. The previously uncharacterized SboB belongs to the type AII class of lantibiotic bacteriocins and is encoded by an eight-gene cluster. The genetic loci encoding SalA2 and SboB in strain K12 have been fully characterized and are localized to nearly adjacent sites on pSsal-K12, a 190-kb megaplasmid. Of 61 strongly inhibitory strains of
S. salivarius
, 19 (31%) were positive for the
sboB
structural gene. All but one (strain NR) of these 19 strains were also positive for
salA2
, and in each of these cases of double positivity, the two loci were separated by fewer than 10 kb. This is the first report of a single streptococcus strain producing two distinct lantibiotics.
Two Complex, Adenovirus-Based Vaccines That Together Induce Immune Responses to All Four Dengue Virus Serotypes
Authors: Holman David H. , Wang Danher , Raviprakash Kanakatte , Raja Nicholas U. , Luo Min , Zhang Jianghui , Porter Kevin R. , Dong John Y.
Published: December 2006

Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.
Experimental
Fasciola hepatica
Infection Alters Responses to Tests Used for Diagnosis of Bovine Tuberculosis
Authors: Flynn Robin J. , Mannion Celine , Golden Olwen , Hacariz Orcun , Mulcahy Grace
Published: December 2006

Fasciola hepatica
is a prevalent helminth parasite of livestock. Infection results in polarization of the host's immune response and generation of type 2 helper (Th2) immune responses, which are known to be inhibitory to Th1 responses. Bovine tuberculosis (BTB) is a bacterial disease of economic and zoonotic importance. Control polices for this disease rely on extensive annual testing and a test-and-slaughter policy. The correct diagnosis of BTB relies on cell-mediated immune responses. We established a model of coinfection of
F. hepatica
and
Mycobacterium bovis
BCG to examine the impact of helminth infection on correct diagnosis. We found the predictive capacity of tests to be compromised in coinfected animals and that
F. hepatica
infection altered macrophage function. Interleukin-4 and gamma interferon expression in whole-blood lymphocytes restimulated in vitro with
M. bovis
antigen was also altered in coinfected animals. These results raise the question of whether
F. hepatica
infection can affect the predictive capacity of tests for the diagnosis of BTB and possibly also influence susceptibility to BTB and other bacterial diseases. Further studies on the interplay between helminth infection and BTB are warranted.
Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA ofListeria monocytogenes
Authors: Mertins Sonja , Joseph Biju , Goetz Monika , Ecke Regina , Seidel Gerald , Sprehe Mareen , Hillen Wolfgang , Goebel Werner , Müller-Altrock Stefanie
Published: December 2006

Analysis ofListeria monocytogenes ptsH,hprK, andccpAmutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. ThehprKmutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in theccpAmutant compared to the wild-type strain. TheptsHmutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in thehprKmutant. As expected, HPr-Ser-P was not produced in thehprKandptsHmutants and synthesized at a similar level in theccpAmutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity whenL. monocytogeneswas grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of thehprKandccpAmutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in thehprKmutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.
Regulation of Rugosity and Biofilm Formation in
Vibrio cholerae
: Comparison of VpsT and VpsR Regulons and Epistasis Analysis of
vpsT
,
vpsR
, and
hapR
Authors: Beyhan Sinem , Bilecen Kivanc , Salama Sofie R. , Casper-Lindley Catharina , Yildiz Fitnat H.
Published: December 2006

Vibrio cholerae
undergoes phenotypic variation that generates two
morphologically different variants, termed smooth and rugose. The transcriptional profiles of the two variants differ greatly, and many of the differentially regulated genes are controlled by a complex regulatory circuitry that includes the transcriptional regulators VpsR, VpsT, and HapR. In this study, we identified the VpsT regulon and
compared the VpsT and VpsR regulons to elucidate the contribution of each positive regulator to the rugose variant transcriptional profile and associated phenotypes. We have found that although the VpsT and VpsR regulons are very similar, the magnitude of the gene regulation accomplished by each regulator is different. We also determined that
cdgA
, which encodes a GGDEF domain protein, is partially responsible for the altered
vps
gene expression between the
vpsT
and
vpsR
mutants. Analysis of epistatic
relationships among
hapR
,
vpsT
, and
vpsR
with respect to a whole-genome expression profile, colony morphology, and biofilm formation revealed that
vpsR
is epistatic to
hapR
and
vpsT
. Expression of virulence genes was increased in a
vpsR hapR
double mutant relative to a
hapR
mutant, suggesting that VpsR negatively regulates
virulence gene expression in the
hapR
mutant. These results show that a complex regulatory interplay among VpsT, VpsR, HapR, and GGDEF/EAL family proteins controls transcription of the genes required for
Vibrio
polysaccharide and virulence factor production in
V.
cholerae
.
Role for the Nonessential N Terminus of FtsN in Divisome Assembly
Authors: Goehring Nathan W. , Robichon Carine , Beckwith Jon
Published: December 2006

FtsN, the last essential protein in the cell division localization hierarchy in
Escherichia coli
, has several peculiar characteristics, suggesting that it has a unique role in the division process despite the fact that it is conserved in only a subset of bacteria. In addition to suppressing temperature-sensitive mutations in
ftsA
,
ftsK
,
ftsQ
, and
ftsI
, overexpression of FtsN can compensate for a complete lack of FtsK in the cell. We examined the requirements for this phenomenon. We found that the N-terminal terminal region (cytoplasmic and transmembrane domains) is critical for suppression, while the C-terminal murein-binding domain is dispensable. Our results further suggest that FtsN and FtsK act cooperatively to stabilize the divisome.
Genomic Structure and Phylogeny of the Plant Pathogen
Ralstonia solanacearum
Inferred from Gene Distribution Analysis
Authors: Guidot Alice , Prior Philippe , Schoenfeld Jens , Carrère Sébastien , Genin Stéphane , Boucher Christian
Published: December 2006

In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic β-proteobacterium
Ralstonia solanacearum
, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the
R. solanacearum
species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the
R. solanacearum
species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that
R. solanacearum
is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.
Cloning and Expression of the
Erwinia carotovora
subsp.
carotovora
Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1
Authors: Chuang Duen-yau , Chien Yung-chei , Wu Huang-Pin
Published: December 2006

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of
Erwinia carotovora
. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn
5
insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of
Erwinia carotovora
subsp.
carotovora
89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn
5
insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes,
caroS1K
(ORF2) and
caroS1I
(ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in
E. carotovora
subsp.
carotovora
Ea1068 and replicated in TH22-10 but could not be expressed in
Escherichia coli
(JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the
E. coli
cyclic AMP receptor protein binding site (−312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in
Pseudomonas aeruginosa
. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.
Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from
Streptococcus mitis
Authors: Jefferies Johanna , Nieminen Leena , Kirkham Lea-Ann , Johnston Calum , Smith Andrew , Mitchell Tim J.
Published: December 2006

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of
Streptococcus mitis
isolates. We have sequenced the mitilysin gene from seven isolates of
S. mitis
. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions.
S. mitis
appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of
S. mitis
may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between
S. mitis
and
Streptococcus pneumoniae
, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.
Kinetic Analysis of the Assembly of the Outer Membrane Protein LamB in
Escherichia coli
Mutants Each Lacking a Secretion or Targeting Factor in a Different Cellular Compartment
Authors: Ureta Alejandro R. , Endres Robert G. , Wingreen Ned S. , Silhavy Thomas J.
Published: December 2006

Outer membrane β-barrel proteins in gram-negative bacteria, such as
Escherichia coli
, must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different subassembly reactions. This kinetic analysis revealed that
secB
and
secD
mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SecD in LamB assembly. Moreover, we found that
surA
mutants are qualitatively indistinguishable from
yfgL
mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.
Cys303 in the Histidine Kinase PhoR Is Crucial for the Phosphotransfer Reaction in the PhoPR Two-Component System in
Bacillus subtilis
Authors: Eldakak Amr , Hulett F. Marion
Published: December 2006

The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the
resA
promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR
C303A
, exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR
C303A
and DTT are different. PhoR and PhoR
C303A
were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoR∼P, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoR∼P or PhoR
C303A
∼P and PhoP occurred rapidly; most label from PhoR∼P was transferred to PhoP, but only 10% of the label from PhoR
C303A
∼P was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoP∼P or PhoR autophosphatase activity was observed between PhoR and PhoR
C303A
that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR
C303
in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoR∼P to PhoP, which is stabilization that is required for efficient production of PhoP∼P.
CC1, a Novel Crenarchaeal DNA Binding Protein
Authors: Luo Xiao , Schwarz-Linek Uli , Botting Catherine H. , Hensel Reinhard , Siebers Bettina , White Malcolm F.
Published: December 2006

The genomes of the related crenarchaea
Pyrobaculum aerophilum
and
Thermoproteus tenax
lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of
T. tenax
. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the
P. aerophilum
and
Aeropyrum pernix
genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in
T. tenax
. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.
Biosynthesis of Phosphoserine in the
Methanococcales
Authors: Helgadóttir Sunna , Rosas-Sandoval Guillermina , Söll Dieter , Graham David E.
Published: December 2006

Methanococcus maripaludis
and
Methanocaldococcus jannaschii
produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA
Cys
with
l
-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in
Methanococcales
, cell extracts of
M. maripaludis
were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from
M. maripaludis
had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from
M. jannaschii
converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The
M. maripaludis
homolog complemented a
serC
mutation in the
Escherichia coli
phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.
Characterization ofHelicobacter pyloriLytic Transglycosylases Slt and MltD
Authors: Chaput Catherine , Labigne Agnès , Boneca Ivo G.
Published: December 2006

Peptidoglycan (PG) is a cell wall heteropolymer that is essential for cell integrity. PG hydrolases participate in correct assembly of the PG layer and have been shown to be required for cell division, cell daughter separation, and maintenance of bacterial morphology. In silico analysis of theHelicobacter pylorigenome resulted in identification of three potential hydrolases, Slt, MltD, and AmiA. This study was aimed at determining the roles of the putative lytic transglycosylases, Slt and MltD, inH. pylorimorphology, growth, and PG metabolism. Strain 26695 single mutants were constructed using a nonpolar kanamycin cassette. ThesltandmltDmutants formed normal bacillary and coccoid bacteria in the exponential and stationary phases, respectively. ThesltandmltDmutants had growth rates comparable to the growth rate of the parental strain. However, themltDmutant exhibited enhanced survival in the stationary phase compared to the wild type or thesltmutant. PG was purified from exponentially growing bacteria and from bacteria in the stationary phase, and its muropeptide composition was analyzed by high-pressure liquid chromatography. This analysis revealed changes in the muropeptide composition indicating that MltD and Slt have lytic transglycosylase activities. Glycan strand analysis suggested that Slt and MltD have exo and endo types of lytic transglycosylase activity, indicating that Slt is involved mainly in PG turnover and MltD is involved mainly in rearrangement of the PG layer. In this study, we determined the distinct roles of the lytic transglycosylases Slt and MltD in PG metabolism.