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.

Phase control of gain and dispersion in an open lambda-type inversionless lasing system

The control role of the relative phase between the probe and driving fields on the gain and dispersion in an open Lambda-type inversionless lasing system with spontaneously generated coherence (SGC) is investigated. It is shown that the inversionless gain and dispersion are quite sensitive to variation in the relative phase; by adjusting the value of the relative phase, electromagnetically induced transparency (EIT), a high refractive index with zero absorption and a larger inversionless gain can be realized. It is also shown that, in the contributions to the inversionless gain ( absorption) and dispersion, the contribution from SGC is always much larger than that from the dynamically induced coherence for any value of the relative phase. Our analysis shows that variation in the SGC effect will cause the spectrum regions and values of the inversionless gain and dispersion to vary evidently. We also found that, under the same conditions, the values of the inversionless gain and dispersion in the open system are evidently larger than those in the corresponding closed system; EIT occurs in the open system but cannot occur in the closed system.

Propagation of a few-cycle laser pulse in a V-type three-level system

Propagation of a few-cycle laser pulse in a V-type three-level system (fine structure levels of rubidium) is investigated numerically. The full three-level Maxwell-Bloch equations without the rotating wave approximation and the standing slowly varying envelope approximation are solved by using a finite-difference time-domain method. It is shown that, when the usual unequal oscillator strengths are considered, self-induced transparency cannot be recovered and higher spectral components can be produced even for small-area pulses. (c) 2005 Pleiades Publishing, Inc.

Six-wave mixing phase-dispersion by optical heterodyne detection in dressed reverse N-type four-level system

We have investigated the dressed effects of non-degenerate four-wave mixing (NDFWM) and demonstrated a phase-sensitive method of studying the fifth-order nonlinear susceptibility due to atomic coherence in RN-type four-level system. In the presence of a strong coupling field, NDFWM spectrum exhibits Autler-Townes splitting, accompanied by either suppression or enhancement of the NDFWM signal, which is directly related to the competition between the absorption and dispersion contributions. The heterodyne-detected nonlinear absorption and dispersion of six-wave mixing signal in the RN-type system show that the hybrid radiation-matter detuning damping oscillation is in the THz range and can be controlled and modified through the colour-locked correlation of twin noisy fields.

Intersubband absorption energy shifts in 3-level system for asymmetric quantum well terahertz emitters

Intersubband absorption energy shifts in 3-level system stemming from depolarization and excitonlike effects are investigated. Analytically, the expressions we derive present good explanations to the conventional 2-level results and bare potential transition energy results; and numerical results show that they are more exact than the previous studies to describe the 3-level system depolarization and excitonlike shift (DES) character especially for higher carrier density (more than 8 x 10(11) cm(-2)). One interesting detail we find is that the "large blue" DES becomes "slight redshift" in the low doping limit (less than 1.9 x 10(11) cm(-2)), which may be neglected by the previous studies of intersubband transitions. Temperature character of DES in the step well structure is also numerically studied. Finally the above are applied to calculate asymmetric step quantum well structures. The two main functional aspects of terahertz (THz) emitters are discussed and several basic optimizing conditions are considered. By adjusting the well geometry parameters and material composition systematically, some optimized structures which satisfy all of the six conditions are recommended in tables. These optimizations may provide useful references to the design of 3-level-based optically pumping THz emitters.

Synthesis of Liquid Polybutadiene Having Desired 1,2-Vinyl Content with Co(naph)(2)-Al-2(C2H5)(3)Cl-3-P(OPh)(3) Catalyst System

Liquid polybutadiene with desirable 1,2-units content was synthesized by Co(naph)(2)-Al-2(C2H5)(3)Cl-3-P(OPh)(3) catalyst system. It was shown that liquid polybutadiene having adequate 1,2-unit content (vinvl =35%-40%) molecular weight(M-n = 700-3500), and acceptabele conversion(>= 55%) can synthesized after optimizing polymerization conditions.

The polymerization of methyl methacrylate with a new Nd(O-i-Pr)(3)-Al(i-Bu)(3) catalyst system

Methyl methacrylate (MMA) was polymerized with the rare earth coordination catalyst-system of Nd(O - i-Pr)(3) in toluene. The influences of various ligands in neodymium complexes, molar ratio of Al/Nd, catalyst concentration, catalyst aging time, solvents, the third component CCl4, temperature and time on the polymerization of MMA were studied. The results showed that the polymerization conversion reached more than 80% at a catalyst concentration of 9.2 x 10(-3) mol/L. The appropriate molar ratio of CCl4/Nd was 4. Hydrocarbon was preferred for the polymerzation to obtain a high conversion and a high <(M)over bar w> of PMMA. The H-1 NMR spectra of PMMA indicated that the lower the temperature, the higher the syndiotactic content of PMMA was obtained.

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.

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.

Phase control of gain and dispersion in an open V-type system

The control role of the relative phase between the probe and driving fields on gain, dispersion and populations in an open V-type three-level system with spontaneously generated coherence is studied. The result shows that by adjusting the value of the relative phase, the transformation between lasing with inversion and lasing without inversion (LWI) can be realized and high dispersion (refractive index) without absorption can be obtained. The shape and value range of the dispersion curve are similar to those of the gain curve, and this similarity is closely related to the relative phase. The effects of the atomic exit and injection rates and the incoherent pump rate on the control role of the relative phase are also analysed. It is found easier to get LWI by adjusting the value of the relative phase using the open system rather than the closed system, and using an incoherent pump rather than without using the incoherent pump. Moreover the open system can give a larger LWI gain than the closed system.

Dressed multi-wave mixing in a V-type four-level atomic system

The dressed four- and six-wave mixings in a V-type four-level system are considered. Under two different dressed conditions, two- and three-photon resonant Autler-Townes splittings, accompanied by enhancement and suppression of wave mixing signal, are obtained analytically. Meanwhile, an electromagnetic induced transparency of multi-wave mixing is presented, which shows multiple peaks and asymmetric effects caused by one-photon, two-photon and three-photon resonances, separately. The slow light propagation multiple region of multi-wave mixing signal is also obtained.

A multi-standard active-RC filter with accurate tuning system

A low-power, highly linear, multi-standard, active-RC filter with an accurate and novel tuning architec-ture is presented. It exhibits 1EEE 802. 11a/b/g (9.5 MHz) and DVB-H (3 MHz, 4 MHz) application. The filter exploits digitally-controlled polysilicon resistor banks and a phase lock loop type automatic tuning system. The novel and complex automatic frequency calibration scheme provides better than 4 comer frequency accuracy, and it can be powered down after calibration to save power and avoid digital signal interference. The filter achieves OIP3 of 26 dBm and the measured group delay variation of the receiver filter is 50 ns (WLAN mode). Its dissipation is 3.4 mA in RX mode and 2.3 mA (only for one path) in TX mode from a 2.85 V supply. The dissipation of calibration consumes 2 mA. The circuit has been fabricated in a 0.35μm 47 GHz SiGe BiCMOS technology; the receiver and transmitter filter occupy 0.21 mm~2 and 0.11 mm~2 (calibration circuit excluded), respectively.

Nanoscale-enhanced Ru(bpy)(3)(2+) electrochemiluminescence labels and related aptamer-based biosensing system

A unique multilabeling at a single-site protocol of the Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system is proposed. Nanoparticles (NPs) were used as assembly substrates to enrich ECL co-reactants of Ru(bpy)(3)(2+) to construct nanoscale-enhanced ECL labels. Two different kinds of NP substrates [including semiconductor NPs (CdTe) and noble metal NPs (gold)] capped with 2-(dimethylamino)ethanethiol (DMAET) [a tertiary amine derivative which is believed to be one of the most efficient of co-reactants of the Ru(bpy)(3)(2+) system] were synthesized through a simple one-pot synthesis method in aqueous media.