Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Analysis of the effect of copper on the virulence of a pathogenic Edwardsiella tarda strain

Aim: To investigate the effect of copper on the virulence of Edwardsiella tarda. Methods and Results: The pathogenic Edw. tarda strain TX5 was cultured under copper-stressed conditions and examined for any potential alteration in capacities that are associated with pathogenicity. The results showed that compared to untreated TX5, Cu-treated TX5 exhibits reduced planktonic and biofilm growth, an impaired ability to adhere to host mucus, modulation of host immune response, and dissemination in host blood and liver. Consistent with these observations, the overall bacterial virulence of Cu-treated TX5 is significantly attenuated. SDS-PAGE analyses of whole cell protein production showed that Cu-treated TX5 differs from the untreated TX5 in its production of at least one protein. Quantitative real time reverse transcriptase PCR analyses showed that copper treatment decreased the expression of virulence-associated genes encoding components of the type III and type VI secretion systems, the Eth haemolysin system, and the LuxS/AI-2 quorum-sensing system. Conclusions: Prolonged exposure to copper has multiple effects on TX5 and results in significant attenuation of bacterial virulence. Significance and Impact of the Study: The results of this study demonstrate that copper treatment has a broad and profound effect on the virulence-associated capacities of TX5, which is exerted at least in part at the transcription level. These findings provide new insights to the antimicrobial mechanism of copper.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

REE and epsilon(Nd) of clay fractions in sediments from the eastern Pacific Ocean: Evidence for clay sources

Clay fractions in the non-calcareous surface sediments from the eastern Pacific were analyzed for clay minerals, REE and Nd-143/Nd-144. Montmorillonite/illite ratio (M/I ratio), total REE contents (Sigma REE), LREE/HREE ratio and cerium anomaly (delta Ce) may effectively indicate the genesis of clay minerals. Clay fractions with M/I ratio > 1, delta Ce < 0.85, Sigma REE > 400 mu g/g, LREE/HREE ratio approximate to 4, and REE patterns similar to those of pelagic sediments are terrigenous and autogenetic mixed clay fractions and contain more autogenetic montmorillonite. Clay fractions with M/I ratio < 1, delta Ce=0.86 to 1.5, Sigma REE=200 to 350 mu g/g, LREE/HREE ratio approximate to 6 and REE distribution patterns similar to that of China loess are identified as terrigenous clay fraction. The Nd-143/Nd-144 ratios or epsilon(Nd) values of clay fractions inherit the features of terrigenous sources of clay minerals. Clay fractions are divided into 4 types according to epsilon(Nd) values. Terrigenous clay minerals of type I with the eNd values of -8 to -6 originate mainly from North American fluvial deposits. Those of type 11 with the epsilon(Nd) Values of -9 to -7 are mainly from the East Asia and North American fluvial deposits. Those of type III with epsilon(Nd) values of -6 to -3 could come from the central and eastern Pacific volcanic islands. Those of type IV with epsilon(Nd) values of -13 to -12 may be from East Asia eolian. The terrigenous and autogenetic mixed clay fractions show patchy distributions, indicating that there are volcanic or hot-spot activities in the eastern Pacific plate, while the terrigenous clay fractions cover a large part of the study area, proving that the terrigenous clay minerals are dominant in the eastern Pacific.

Dynamics of the buoyant plume off the Pearl River Estuary in summer

Field measurements of salinity, wind and river discharge and numerical simulations of hydrodynamics from 1978 to 1984 are used to investigate the dynamics of the buoyant plume off the Pearl River Estuary (PRE), China during summer. The studies have shown that there are four major horizontal buoyant plume types in summer: Offshore Bulge Spreading (Type I), West Alongshore Spreading (Type II), East Offshore Spreading (Type III), and Symmetrical Alongshore Spreading (Type IV). River mouth conditions, winds and ambient coastal currents have inter-influences to the transport processes of the buoyant plume. It is found that all of the four types are surface-advected plumes by analysing the vertical characteristic of the plumes, and the monthly variations of the river discharge affect the plume size dominantly. The correlation coefficient between the PRE plume size and the river discharge reaches 0.85 during the high river discharge season. A wind strength index has been introduced to examine the wind effect. It is confirmed that winds play a significant role in forming the plume morphology. The alongshore wind stress and the coastal currents determine the alongshore plume spreading. The impact of the ambient currents such as Dongsha Current and South China Sea (SCS) Warm Current on the plume off the shelf has also assessed. The present study has demonstrated that both the river discharge and wind conditions affect the plume evolution.

AGVS系统技术应用

本文在分析装配型ＡＧＶＳ系统和运输型ＡＧＶＳ系统的基础上，详细说明ＡＧＶＳ主要技术指标，性能特点和应用原则。同时，结合分析自动导引车的发展历史，说明自动导引车系统的应用领域，分析推测自动导引车的发展前景。

Low-Dimensional Dynamical Systems for a Wake-Type Shear Flow with Vortex Dislocations

Low-dimensional systems are constructed to investigate dynamics of vortex dislocations in a wake-type shear flow. High-resolution direct numerical simulations are employed to obtain flow snapshots from which the most energetic modes are extracted using proper orthogonal decomposition (POD). The first 10 modes are classified into two groups. One represents the general characteristics of two-dimensional wake-type shear flow, and the other is related to the three-dimensional properties or non-uniform characteristics along the span. Vortex dislocations are generated by these two kinds of coherent structures. The results from the first 20 three-dimensional POD modes show that the low- dimensional systems have captured the basic properties of the wake-type shear flow with vortex dislocation, such as two incommensurable frequencies and their beat frequency.

Phthalocyanato tin(IV) dichloride: An air-stable, high-performance, n-type organic semiconductor with a high field-effect electron mobility

Phthalocyanato tin(IV) dichloride, an axially dichloriniated MPc, is an air-stable high performance n-type organic semiconductor with a field-effect electron mobility of up to 0.30 cm(2) V-1 s(-1). This high mobility together with good device stability and commercial availability makes it a most suitable n-type material for future organic thin-film transistor applications.

Interfacial behavior of primary amine N1923 and the kinetics of thorium(IV) extraction in sulfate media

The influences of additive, diluents, temperature, acidity of the aqueous phase on the interfacial behavior of primary amine N1923 in sulfate media have been investigated using the Du Nouy ring method. In addition, the effect of concentration of thorium(IV) loaded in the organic phase on the interfacial tension has also been studied. The interfacial tension isotherms are processed by matching different adsorption equations such as the Gibbs and the Szyszkowski. The surface excess at the saturated interface (Gamma (max)) and the minimum bulk concentration of the extractant necessary to saturate the interface (C-min) under different conditions are calculated according to two adsorption equations to be presented in comprehensive tables and figures. It appears that primary amine N1923 has strong interfacial activity and behaves very differently in various diluents systems. The surface excess at saturated interface increase with the type of diluerits in the following order: chloroform < aromatic hydrocarbons < aliphatic hydrocarbons. The relationship between the interfacial activity and kinetics of thorium extraction by primary amine N1923 has been discussed by considering different factors. However, the interfacial activity of primary amine N1923 is only a qualitative parameter suggesting the interfacial mechanism for thorium extraction, it cannot give strong evidence quantitatively supporting this mechanism.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Creation of arbitrary coherent superposition states in four-level systems

Using the technique of stimulated Raman adiabatic passage, we propose schemes for creating arbi- trary coherent superposition states of atoms in four-level systems: a A-type system with twofold final states and a four-level ladder system. With the use of a control field, arbitrary coherent superposition states are created without the condition of multiphoton resonance. Suitable manipulation of detunings and the control field can create either a single state or any superposition states desired. (c) 2005 Pleiades Publishing, Inc.