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1.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836580

RESUMO

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Mimetismo Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mycobacterium/enzimologia , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Clivagem do DNA , Proteínas Monoméricas de Ligação ao GTP/química , Conformação Proteica
2.
Opt Express ; 31(8): 13455-13471, 2023 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-37157484

RESUMO

The practical Kramers-Kronig (KK) receiver has been a competitive receiving technique in the data-center, medium reach, and even long-haul metropolitan networks. Nevertheless, an extra digital resampling operation is required at both ends of the KK field reconstruction algorithm due to the spectrum broadening caused by adopting the nonlinear function. Generally, the digital resampling function can be implemented by using linear interpolation (LI-ITP), the Lagrange cubic interpolation (LC-ITP), the spline cubic interpolation (SC-ITP), time-domain anti-aliasing finite impulse response (FIR) filter method (TD-FRM) scheme, and fast Fourier transform (FFT)-based scheme. However, the performance and the computational complexity analysis of different resampling interpolation schemes in the KK receiver have not been thoroughly investigated yet. Different from the interpolation schemes of conventional coherent detection, the interpolation function of the KK system is followed by the nonlinear operation, which will broaden the spectrum significantly. Due to the frequency-domain transfer function of different interpolation schemes, the broadened spectrum will have a potential spectrum aliasing, which will cause serious inter-symbol interference (ISI) and further impair the KK phase retrieval performance. We experimentally investigate the performance of different interpolation schemes under different digital up-sampling rates (i.e. the computational complexity) as well as the cut-off frequency, the tap number of the anti-aliasing filter, and the shape factor of the TD-FRM scheme in a 112-Gbit/s SSB DD 16-QAM system over 1920-km Raman amplification (RFA)-based standard single-mode fiber (SSMF). The experimental results involve that the TD-FRM scheme outperforms other interpolation schemes and the complexity is reduced by at least 49.6%. In fiber transmission results, take 20% soft decision-forward error correction (SD-FEC) of 2×10-2 as the threshold, the LI-ITP and LC-ITP schemes only reach 720-km while others can reach up to 1440-km.

3.
Appl Opt ; 62(17): 4482-4489, 2023 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-37707140

RESUMO

A few-mode erbium-doped fiber (FM-EDF) with a step refractive index and trench structure is designed and proposed to realize the modal gain equalization of a few-mode erbium-doped fiber amplifier (FM-EDFA). The layered-doping technology is used to reduce the mode gain difference (DMG). The doping radius and doping concentration are adjusted to obtain the optimum FM-EDF structure. When the designed FM-EDF is applied to the FM-EDFA, the DMG of the whole C-band is less than 0.15 dB and the DMG is less than 0.12 dB at 1550 nm. The minimum refractive index difference (Δ n eff) between modes can be calculated according to the refractive index and radius of the fiber core; i.e., 1.35×10-3, which will greatly reduce the coupling between modes in a practical application. Tolerances in the fiber manufacturing process are also considered for reliable FM-EDFA performance. When the doping radius and concentration of each doping layer fluctuate by ±15% based on the precise value, the maximum DMG increases to 1.8 dB. In general, DMG can maintain a small value, which is beneficial for application in optical communications systems.

4.
Opt Express ; 27(9): 13150-13159, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31052844

RESUMO

We propose and experimentally demonstrate a novel scheme to generate LP11/LP21 modes with tunable mode lobe orientation (MLO). Wherein, the MLOs have an excellent linear relationship with the linearly-polarized states of input fundamental modes. The proposed scheme is composed of a polarization controller (PC), a mode converter, a mode and polarization controller (PMC) which is twined with the few mode fiber (FMF) and a polarizer. Experimental results show that the deviations of MLOs between generated LP11/LP21 modes and simulated ones are less than 3.5 and 8 degrees over C band. Since polarization control up to nanosecond scale is available with GaAs or lithium based electro-optic modulator, the proposed scheme could enable nanosecond time scale MLO control, which would be immensely useful for optical trapping, fiber sensors and optical communications.

5.
Opt Express ; 27(24): 35537-35547, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31878723

RESUMO

Based on the spatial profiles and polarization states evolution process of the first-order modes resulted from stress-induced birefringence in the few-mode fiber (FMF), we analyze the mapping relationship between the input polarization states represented on polarization PS and the output spatial profiles represented on the orbital PS of the FMF with respect to the magnitude and orientation of birefringence. When the input mode lobe orientation and the phase differences between the four eigenmodes of FMF induced by the stress birefringence satisfy a given condition, the mapping relationship between the input polarization PS and the output orbital PS is linear. Thus, the arbitrary points on the orbit PS can be generated at the output of stressed FMF by controlling the polarization state of the input modes. Then we experimentally verify that, an electrical single-mode polarization controller, a mode converter for converting fundamental mode to higher-order mode, a polarization controller mounting a coil of two-mode fiber and a polarizer can be employed to generate arbitrary first-order spatial modes on the orbital PS by controlling the input single-mode polarization states. The positions on the orbital PS of the generated first-order modes, which are obtained by calculating the three normalized Stokes parameters of output modes, agree well with the simulation ones. The correlation coefficients between the theoretical mode profiles and the experimental ones are higher than 80%. Since the spatial profile evolutions depend on the variations of the input polarization states, a potential advantage of this method is high-speed switching among desired first-order modes by using the commercial devices switching the state of polarization.

6.
J Bacteriol ; 200(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29378889

RESUMO

During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/metabolismo , Concentração de Íons de Hidrogênio , Mutação , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidade , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Fosforilação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
J Bacteriol ; 200(12)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29632095

RESUMO

The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the ß' subunit (ß'CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this ß'CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis Sequence alignment of the ω loop and the ß'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly.IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α2ßß'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis, and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium tuberculosis/metabolismo , Fator sigma/química , Fator sigma/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Ligação Proteica , Fator sigma/genética , Transcrição Gênica
8.
Opt Lett ; 43(19): 4779-4782, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30272738

RESUMO

We demonstrate for the first time, to the best of our knowledge, an all-fiber orbital angular momentum (OAM) multiplexer that multiplexes both OAM modes of -l and +l up to the second order by using a mode-selective photonic lantern and a mode polarization controller. The experimentally obtained mode profiles are close to the theoretical results, and the mode purities are higher than 89% for all the OAM modes at 1550 nm. The losses for all mode generations are less than 3.8 dB in the C-band.

9.
Opt Lett ; 43(9): 2130-2133, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29714763

RESUMO

In this Letter, we theoretically and experimentally demonstrate a new method to generate tunable orbital angular momentum (OAM) by continuously changing the angle of linear polarization of the input light. We use the Fourier series of left- and right-hand projections to prove that the average OAM smoothly varied from l=-1 to l=1 with the angle of LP of input light changing from 0 to π, which is fulfilled by an electrical polarization controller.

10.
Appl Opt ; 57(36): 10528-10533, 2018 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-30645400

RESUMO

This paper proposes and experimentally demonstrates an all-fiber conversion method that transfers the polarization-division multiplexing (PDM) signals to the mode-division multiplexing (MDM) signals. The conversion scheme is based on a mode converter and a polarization-mode controller. The input X-polarized/Y-polarized fundamental modes are converted to the first-order linear-polarized LP11A/LP11B modes with crosstalk of -10 dB/-18 dB and insertion losses of 3.04 dB/3.1 dB at 1550 nm, respectively. Using the proposed converter, 11.2 GBaud/s polarization-division-multiplexed quadrature-phase-shift keying (PDM-QPSK) and 16 quadrature-amplitude-modulation (PDM-16QAM) signals are successfully converted to 11.2 GBaud/s MDM-QPSK and MDM-16QAM signals.

11.
Mol Microbiol ; 102(1): 107-20, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27353316

RESUMO

Mycobacterium tuberculosis adopts various strategies to cope with oxidative stress during infection. Transcriptional regulators, including σ factors, make important contributions to this stress response, but how these proteins cooperate with each other is largely unknown. In this study, the role of RbpA and its cooperation with σ factors in response to oxidative stress are investigated. Knock down expression of rbpA in Mycobacterium smegmatis attenuated bacterial survival in the presence of H2 O2 . Additionally, transcription of the rbpA gene was induced by H2 O2 in a σ(E) -dependent manner. After induction, RbpA interacts with the principal sigma factor, σ(A) , to control the transcription of furA-katG operon, which encodes an H2 O2 scavenging enzyme. Moreover, this regulation is responsible for the role of σ(E) in oxidative response because bacterial survival was attenuated and transcription of the furA-katG operon was down-regulated with H2 O2 treatment in sigE deletion mutant (ΔsigE), and over-expression of RbpA in ΔsigE strain restored all of these phenotypes. Taken together, our study first illustrated a mechanism for σ(E) in response to oxidative stress through regulation of rbpA transcription. This study was also the first to demonstrate that RbpA is required for the full response to oxidative stress by cooperating with the principal σ(A) .


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Regiões Promotoras Genéticas , Fator sigma/genética , Transcrição Gênica
12.
Microbiology (Reading) ; 162(5): 889-897, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26887897

RESUMO

Gene transcription catalysed by RNA polymerase is regulated by transcriptional regulators, which play central roles in the control of gene transcription in both eukaryotes and prokaryotes. In regulating gene transcription, many regulators form dimers that bind to DNA with repeated motifs. However, some regulators function as monomers, but their mechanisms of gene expression control are largely uncharacterized. Here we systematically characterized monomeric versus dimeric regulators in the tuberculosis causative agent Mycobacterium tuberculosis. Of the >160 transcriptional regulators annotated in M. tuberculosis, 154 transcriptional regulators were tested, 22 % probably act as monomers and most are annotated as hypothetical regulators. Notably, all members of the WhiB-like protein family are classified as monomers. To further investigate mechanisms of monomeric regulators, we analysed the actions of these WhiB proteins and found that the majority interact with the principal sigma factor σA, which is also a monomeric protein within the RNA polymerase holoenzyme. Taken together, our study for the first time globally classified monomeric regulators in M. tuberculosis and suggested a mechanism for monomeric regulators in controlling gene transcription through interacting with monomeric sigma factors.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium tuberculosis/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Multimerização Proteica/genética , Transcrição Gênica/genética
13.
Zhongguo Zhong Yao Za Zhi ; 40(20): 4112-6, 2015 Oct.
Artigo em Zh | MEDLINE | ID: mdl-27062835

RESUMO

A complex disease is rarely a consequence of abnormality in a single gene. It is known that many drugs exhibit a therapeutic effect by acting on multiple targets, produce synergies to intervene the occurrence and development of diseases. Unlike the traditional methods which act on single molecule or pathway, this disease-drug target network constructed with high throughput data vividly showed the complex relationship between drugs, their targets and diseases. However, the networks are usually extremely complex. In order to reduce the complexity, it is necessary to deconstruct the network and identify module structures. In this study, framework of module analysis was summarized from four aspects: module concept, structure and identification methods, importance of disease-drug module identification, and its application. Module-based analysis provides a new perspective for deciphering the drug intervention mechanisms for complex diseases, and provides new ideas and pathways to reveal the mechanisms of multi-target and multi-component drugs.


Assuntos
Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Terapia de Alvo Molecular , Sistemas de Liberação de Medicamentos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos
14.
Guang Pu Xue Yu Guang Pu Fen Xi ; 33(10): 2603-6, 2013 Oct.
Artigo em Zh | MEDLINE | ID: mdl-24409699

RESUMO

The present paper reports the high resolution transient Raman laser testing technology under two-stage light gas-gun loading experiment, and its application to studying the Raman spectroscopy of shocked benzene. In the experiments, the frequency shift of C-C stretching vibration (992 cm(-1)) and C-H stretching vibration peak (3 061 cm(-1)) in the low pressure section (less than 8 GPa) varies linearly with the pressure, and the results agree well with reported data in the literature. The structural changes in liquid benzene about 13 GPa were clarified firstly by the Raman spectral technique; the experimental results show that at a pressure of 9.7 GPa, the structural change of liquid benzene has taken place, not reported in the literature about 13 GPa. But the composition in the production is not clear. The measurement system provides an effective means to study the microstructure changes of transparent and translucent material under dynamic loading experiment.

15.
RSC Med Chem ; 13(7): 831-839, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35919336

RESUMO

By 2050, it is predicted that antimicrobial resistance will be responsible for 10 million global deaths annually, more deaths than cancer, costing the world economy $100 trillion. Clearly, strategies to address this problem are essential as bacterial evolution is rendering our current antibiotics ineffective. The discovery of an allosteric binding site on the established antibacterial target DNA gyrase offers a new medicinal chemistry strategy. As this site is distinct from the fluoroquinolone binding site, resistance is not yet documented. Using in silico molecular design methods, we have designed and synthesised a novel series of biphenyl-based inhibitors inspired by a published thiophene-based allosteric inhibitor. This series was evaluated in vitro against Escherichia coli DNA gyrase and E. coli topoisomerase IV with the most potent compounds exhibiting IC50 values towards the low micromolar range for DNA gyrase and only ∼2-fold less active against topoisomerase IV. The structure-activity relationships reported herein suggest insights to further exploit this allosteric site, offering a pathway to overcome developing fluoroquinolone resistance.

16.
Cell Rep ; 16(9): 2512-24, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545883

RESUMO

During the course of infection, Mycobacterium tuberculosis (Mtb) is exposed to diverse redox stresses that trigger metabolic and physiological changes. How these stressors are sensed and relayed to the Mtb transcriptional apparatus remains unclear. Here, we provide evidence that WhiB6 differentially regulates the ESX-1 and DosR regulons through its Fe-S cluster. When challenged with NO, WhiB6 continually activates expression of the DosR regulons but regulates ESX-1 expression through initial activation followed by gradual inhibition. Comparative transcriptomic analysis of the holo- and reduced apo-WhiB6 complemented strains confirms these results and also reveals that WhiB6 controls aerobic and anaerobic metabolism, cell division, and virulence. Using the Mycobacterium marinum zebrafish infection model, we find that holo- and apo-WhiB6 modulate levels of mycobacterial infection, granuloma formation, and dissemination. These findings provide fresh insight into the role of WhiB6 in mycobacterial infection, dissemination, and disease development.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Mycobacterium marinum/genética , Regulon , Fatores de Transcrição/genética , Aerobiose/genética , Sequência de Aminoácidos , Anaerobiose/genética , Animais , Proteínas de Bactérias/metabolismo , Doenças dos Peixes/microbiologia , Doenças dos Peixes/patologia , Granuloma/microbiologia , Granuloma/patologia , Granuloma/veterinária , Interações Hospedeiro-Patógeno , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/efeitos dos fármacos , Mycobacterium marinum/crescimento & desenvolvimento , Mycobacterium marinum/patogenicidade , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Transcriptoma , Triazenos/química , Triazenos/farmacologia , Virulência , Peixe-Zebra/microbiologia
17.
PLoS One ; 9(3): e92243, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24658611

RESUMO

Pathogenic Yersinia species employ the Ysc-Yop type III secretion system (T3SS) encoded by a highly conserved pYV virulence plasmid to export the virulence effectors into host cells. The Ysc-Yop T3SS is tightly regulated by multiple contributing proteins that function at different levels. However, systematic transcriptional regulation analysis of Ysc-Yop T3SS is lacking and the detailed mechanism under this regulation process is still elusive. Aimed at systematically characterizing transcriptional regulations of all T3SS genes in Y. pseudotuberculosis, we amplified 97 non-coding fragments from the pYV plasmid and analyzed transcriptional responses of the T3SS genes under different growth conditions. Transcriptions of T3SS genes were induced at 37°C and genes encoding T3SS effectors were highly induced by further depletion of Ca2+. The temperature induced gene transcription process is mediated by modules encoded on the chromosome, while the Ca2+ depletion-induced process is controlled by the positive regulatory protein LcrF as well as the negative regulatory protein LcrQ. In this process, LcrQ shares the same targets with LcrF and the effect of LcrQ is dependent on the presence of LcrF. Furthermore, over-expression of LcrF showed the same phenotype as that of the lcrQ mutant strain and intracellular amount balance of LcrQ and LcrF is important in T3SS regulation. When the expression level of LcrF exceeds LcrQ, expression of the Ysc-Yop T3SS genes is activated and vice versa. Together, these data support a model in which LcrQ blocks the activation role of LcrF in regulating the transcription of T3SS genes in Yersinia.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/fisiologia , Sistemas de Secreção Bacterianos/genética , Regulação Bacteriana da Expressão Gênica , Transativadores/antagonistas & inibidores , Yersinia pseudotuberculosis/genética , Proteínas de Bactérias/genética , Cálcio/farmacologia , Genes Bacterianos , Plasmídeos , Regiões Promotoras Genéticas , Transcrição Gênica , Virulência/genética
18.
PLoS One ; 7(11): e50142, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185557

RESUMO

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Plasmídeos , Ribossomos/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Bases , Sítios de Ligação , Bioensaio , Expressão Gênica , Vetores Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Ribossomos/metabolismo , Transformação Bacteriana
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