Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagic Escherichia coli (EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression of torR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression of glpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.     

Gene Expression and Signal Transduction in Water-Stress Response

We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8-12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.     

农杆菌双组分信号转导系统中VirG蛋白结构与转化功能的分析

农杆菌介导的转化方法在植物、真菌基因转化上有着重要意义,其中VirG蛋白起着重要作用.利用多种序列、结构分析对比软件,对根癌农杆菌和发根农杆菌4种代表性VirG蛋白基本性质、一级结构、二级结构、三级结构和表面静电势进行了预测比较和分析,发现N端差异较大.来自发根农杆菌的VirG蛋白更是在N端缺失了一段氨基酸,这可能是发根农杆菌转化效率低于根癌农杆菌的一个重要原因.结合文献关于定点突变的研究结果,经过总结分析VirG蛋白各位点突变对功能的影响,发现只有诸如N54和I77等少数位点的突变才对功能有促进作用.这些研究结果为提高农杆菌,尤其是发根农杆菌的侵染能力和范围提供了理论依据.     

Two-Component System Cross-Regulation Integrates Bacillus anthracis Response to Heme and Cell Envelope Stress

Two-component signaling systems (TCSs) are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK) to a cytoplasmic response regulator (RR) that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS). HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.     

金黄色葡萄球菌双组分系统反应调节蛋白AgrA的原核表达、纯化及活性鉴定

AgrA作为金黄色葡萄球菌双组分信号转导系统(two-component signal transduction system,TCST)的反应调节因子,能调控细菌毒力因子的表达,在金黄色葡萄球菌致病过程中起着重要的作用。采用无限制克隆法构建AgrA表达载体,在AgrA蛋白的C端融合绿色荧光蛋白(GFP)标签,通过实时监测GFP的荧光强度来快速检测重组蛋白的表达水平。首先利用单因素实验,筛选出宿主菌株BL21-(DE3)-PlysS;其次,结合Box-Behnken试验设计,筛选出最优蛋白质表达条件:诱导时间为22h、转速为222r/min、诱导剂浓度为0.5mmol/L,AgrA产量达到5.56mg/L。最后,基于AgrA蛋白LytTR区域的非放射性凝胶阻滞实验(non-radioactive electrophoretic mobility shift assay,EMSA)验证了AgrA的生物活性。提出了反应调节蛋白AgrA在大肠杆菌高效可溶性表达的策略,为双组分信号转导系统的体外研究奠定基础,也为其他反应调节蛋白的可溶性表达与分离纯化提供了一个可行借鉴。     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C4-Dicarboxylates in Escherichia coli

The dcuB gene of Escherichia coli encodes an anaerobic C₄-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C₄-dicarboxylates, suggesting the presence of a novel C₄-dicarboxylate-responsive regulator in E. coli. This paper describes the isolation of a Tn10 mutant in which the 160-fold induction of dcuB expression by C₄-dicarboxylates is absent. The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at ~93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C₄-dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS⁺ strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C₄-dicarboxylate transport or metabolism. Expression of the fumarate reductase (frdABCD) operon and the aerobic C₄-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C₄-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C₄-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C₄-dicarboxylates anaerobically revealed that C₄-dicarboxylate transport is not required for C₄-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ~140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C₄-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae. DcuS is not closely related to the C₄-dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.     

植物在渗透胁迫下的基因表达及信号传递

渗透胁迫是指由于环境因素的变化使植物不能得到充足水分的一种状况。常见的渗透胁迫因素有干旱、盐害及冻害等。渗透胁迫会严重影响植物的生长发育及产量。植物在长期的进化过程中形成了一些保护机制能减轻渗透胁迫造成的伤害。比如有些植物在形态上演化生成盐腺,能排出体内的过量盐分以逃避盐害。但在渗透胁迫下几乎所有的植物都能合成一些无毒性的小分子有机物作为渗透调节剂来维持细胞内渗透势的相对稳定。在分子水平上...     

Identification and Disruption of lisRK, a Genetic Locus Encoding a Two-Component Signal Transduction System Involved in Stress Tolerance and Virulence in Listeria monocytogenes

lisRK encodes a two-component regulatory system in the food pathogen Listeria monocytogenes LO28. Following identification of the operon in an acid-tolerant Tn917 mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.     

Role of the CpxAR Two-Component Signal Transduction System in Control of Fosfomycin Resistance and Carbon Substrate Uptake

Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.     

Redox Control of Signal Transduction,Gene Expression and Cellular Senescence

Reactive oxygen species (ROS) act as subcellular messengers in such complex cellular processes as mitogenic signal transduction, gene expression, regulation of cell proliferation, replicative senescence, and apoptosis. They serve to maintain cellular homeostasis and their production is under strict control. However, the mechanisms whereby ROS act are still obscure. Here we review recent advances in our understanding of signaling mechanisms and recent data about the involvement of ROS in: (i) the regulation of the mitogenic transduction elements, particularly protein kinases and phosphatases; (ii) the regulation of gene expression; and (iii) the induction of replicative senescence and the role, if any, in aging and age-related disorders.     

The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1_LadS→D1_LadS→H2_GacS→D2_GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.     

磷脂酶D(PLD)基因的结构、表达及其表达产物在信号转导中的作用

磷脂酶D(PLDEC 3 .1 .4.4)水解磷脂 (PL) ,磷脂构成生物膜的骨架 ,磷脂酶的激活不仅对细胞的结构和稳定性有很重要的作用 ,而且调控许多重要的细胞生理功能 ,例如PLD在信号转导、小泡运输、有丝分裂、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用。近年来它在跨膜信号转导中的重要作用 ,越来越引起人们的重视 ,成为新的研究热点。介绍了磷脂酶基因的结构特点、亚细胞定位、表达的激活抑制以及其表达产物作为胞内信号分子在植物信号转导中的重要作用。     

Identification and Expression Analysis of Abscisic Acid Signal Transduction Genes in Hemp Seeds

Abscisic acid (ABA) is involved in regulating diverse biological processes, but its signal transduction genes and roles in hemp seed germination are not well known. Here, the ABA signaling pathway members, PYL, PP2C and SnRK2 gene families, were identified from the hemp reference genome, including 7 CsPYL (pyrab-actin resistance1-like, ABA receptor), 8 CsPP2CA (group A protein phosphatase 2c), and 7 CsSnRK2 (sucrose nonfermenting1-related protein kinase 2). The content of ABA in hemp seeds in germination stage is lower than that in non-germination stage. Exogenous ABA (1 or 10 μM) treatment had a significant regulatory effect on the selected PYL, PP2C, SnRK2 gene families. CsAHG3 and CsHAI1 were most significantly affected by exogenous ABA treatment. Yeast two-hybrid experiments were performed to reveal that CsPYL5, CsSnRK2.2, and CsSnRK2.3 could interact with CsPP2CA7 and demonstrate that this interaction was ABA-independent. Our results indicated that CsPYL5, CsSnRK2.2, CsSnRK2.3 and CsPP2CA7 might involve in the ABA signaling transduction pathway of hemp seeds during the hemp seed germination stages. This study suggested that novel genetic views can be brought into investigation of ABA signaling pathway in hemp seeds and lay the foundation for further exploration of the mechanism of hemp seed germination.