c-di-GMP抑制转录调控因子Clpxoo与葡聚糖酶基因启动子的结合北大核心CSCD

【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)c-di-GMP信号受体和转录调控因子Clpxoo对葡聚糖酶基因(engAxoo)的转录调控作用机制。【方法】通过基因表达载体的构建和转化、蛋白诱导表达及其Ni-NTA Resin亲和层析,进行了clpxoo基因的原核表达和产物纯化。通过荧光素(FAM)探针标记和凝胶阻滞试验(EMSA)对Clpxoo纯化蛋白与葡聚糖酶基因启动子(engAxoo-p)的结合活性及其c-diGMP信号分子的抑制作用进行了测定分析。【结果】在优化的诱导表达和纯化条件下,成功地获得了Clpxoo纯化蛋白。Clpxoo可与engAxoo-p序列发生阻滞现象,表明它具有与启动子特异性结合的活性。在反应体系中加入c-di-GMP可导致结合作用的消除。【结论】Clpxoo接受c-di-GMP信号后,可能通过构象的改变,从而与engAxoo-p的结合活性受到抑制;优化的Clpxoo蛋白纯化和EMSA方法可以用于后续的调控元大规模鉴定的研究。     

短短芽孢杆菌GZDF3中PilZ结构域基因的挖掘和糖基转移酶的原核表达

【背景】PilZ结构域是最早发现的环二鸟苷酸(Cyclic diguanylate,c-di-GMP)受体信号分子,与c-di-GMP结合后可以调控目标基因或者蛋白的活性,在细菌的生长过程中发挥着至关重要的作用,而短短芽孢杆菌中PilZ结构域的研究相对缺乏。【目的】挖掘短短芽孢杆菌GZDF3菌株中的PilZ结构域蛋白基因,并进行重组表达,为研究其功能奠定基础。【方法】从Pfam数据库中下载PilZ结构域模型,HMMScan软件扫描GZDF3全基因组序列,在保守结构域数据库(Conserved domain database,CDD)中分析蛋白保守结构域,Protein BLAST比对分析;采用ExPASy在线软件预测蛋白的基本理化性质;构建重组表达载体进行蛋白重组表达。【结果】GZDF3基因中存在5个含有PilZ结构域的蛋白编码基因,其中命名为Gene4836的基因经Protein BLAST比对分析显示其编码糖基转移酶,Gene1423为YcgR超家族蛋白编码基因,Gene1723编码透明质酸合成酶,属于糖基转移酶超家族2,其余Gene2571、Gene2956编码假定蛋白;Gene4836的编码产物分子量为24.08 kD,等电点为6.39,为酸性亲水性蛋白;C端有一个PilZ结构域;0.5 mmol/L乳糖诱导、30°C培养20 h,表达出一大小约为25kD的重组蛋白,与生物信息学预测结果相符。【结论】首次对短短芽孢杆菌含有PilZ结构域蛋白编码基因进行原核表达,并成功纯化出重组蛋白,为后续研究其功能奠定了基础。     

水稻白叶枯病菌c-di-GMP受体Clpxoo关键结合功能位点的确定

旨在确定水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)c-di-GMP信号受体Clpxoo的关键功能位点。通过对Clpxoo蛋白氨基酸位点基因突变,表达载体构建、蛋白诱导表达及其Ni-NTA Resin亲和层析,进行了Clpxoo及其点突变蛋白的原核表达和产物纯化;通过等温滴定量热法(ITC),检测了Clpxoo及其点突变蛋白与c-di-GMP的结合作用。利用基因定点突变和桥接PCR方法,在优化的诱导表达和纯化条件下,成功地获得了Clpxoo点突变蛋白和与c-di-GMP不发生结合的Clpxoo点突变体。结果表明,Clpxoo蛋白第70位天冬氨酸和第99位谷氨酸是与c-di-GMP结合的关键功能位点。     

乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的拮抗作用

【目的】对嗜酸乳杆菌的S-层蛋白(S-layer protein)进行提纯,研究嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附和入侵的拮抗作用。【方法】应用阴离子交换柱(DE52)对嗜酸乳酸杆菌的S-层蛋白进行提纯,然后分别研究了嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的作用。【结果】S-层蛋白能显著地抑制鼠伤寒沙门氏菌的黏附及入侵;在竞争、排斥、置换3种黏附试验中,S-层蛋白可显著降低鼠伤寒沙门氏菌的黏附,其相对黏附力分别为1.17%±5.97%、8.71%±1.36%、10.56%±0.92%,差异极显著(p0.01),其中竞争试验效果最好;并且S-层蛋白对鼠伤寒沙门氏菌黏附抑制作用极显著高于嗜酸乳酸杆菌(p0.01);此外,S-层蛋白也能显著抑制鼠伤寒沙门氏菌入侵。【结论】乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌可产生显著的拮抗作用,这可能与S-层蛋白和鼠伤寒沙门氏菌的宿主黏附受体存在竞争作用有关;提示乳酸杆菌S-层蛋白可用于预防和治疗鼠伤寒沙门氏菌感染,并有望成为抗生素的替代品。     

快速纯化重组腺联病毒的受体结合捕捉方法

【目的】建立用于重组腺联病毒(AAV)纯化的受体结合捕捉方法。【方法】将AAV受体的多囊肾病(PKD)结构域1和2与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,利用相变循环(ITC)纯化ELP-PKD融合蛋白;分别用昆虫和AAV-293细胞制备rAAV-GFP,与ELP-PKD融合蛋白共孵育后进行ITC,从沉淀复合物提取病毒DNA进行PCR检测;在优化条件下利用ELP-PKD蛋白结合捕捉rAAV-GFP,利用电子显微镜观察、免疫转印和细胞感染试验进行rAAV鉴定。【结果】ELP-PKD融合蛋白获得正确、可溶性表达,ITC纯化的蛋白纯度大于90%;ELP-PKD蛋白能特异结合rAAV-GFP,结合具有p H、温度和时间依赖性,受体结合捕捉方法可在1h内完成,从两种细胞纯化rAAV-GFP的回收率分别为58%和56%;rAAV-GFP洗脱具有p H和温度依赖性,洗脱rAAV-GFP的回收率分别为46%和44%;纯化rAAV-GFP具有AAV的典型形态和结构蛋白。【结论】建立的ELP-PKD结合捕捉法可用于不同细胞源rAAV的快速纯化。     

SAPl8和Sufu蛋白质复合物的表达纯化研究

通过分子克隆技术将Sufu和SAP18构建到原核载体,在原核表达系统进行外源表达,采用亲和层析和分子筛层析对Sufu和SAPl8及其复合物进行纯化。同时利用非变性胶的方法进一步确定该蛋白之间的相互作用及结合比例。结果表明,原核表达系统中Sufu和SAPl8蛋白表达量高,可以纯化到高纯度的蛋白质。su如和SAPl8结合的摩尔比为1：1,按摩尔比1：2混合、纯化可以得到高纯度、稳定的蛋白复合物,从而为进一步复合物的结构生物学研究奠定基础。     

高盐环境下鼠伤寒沙门氏菌差异糖蛋白富集及组学分析

【背景】沙门氏菌是一种革兰阴性肠道病原菌,主要依靠III型分泌系统(type III secretion systems,T3SSs)来产生与致病性相关的效应蛋白。其中沙门氏菌致病岛(Salmonella pathogenicity island,SPI)区域是关键的基因区域。高盐浓度条件可以诱导SPI-1上效应蛋白的表达。【目的】探究在高盐浓度条件下鼠伤寒沙门氏菌(Salmonella enterica serovar Typhimurium)糖蛋白的差异表达情况,寻找有意义的效应糖蛋白。【方法】将鼠伤寒沙门氏菌在普通培养基和高盐培养基中培养,收集菌体并超声裂解,提取蛋白后,用肼偶联法富集糖蛋白并用胰酶酶解,通过二甲基标记定量及LC/MS定量蛋白质组学方法进行糖蛋白的定量,用Thermo Proteome Discoverer 2.2软件对标记蛋白进行定性及定量分析。【结果】质谱结果显示,高盐环境中,沙门氏菌有19个糖蛋白的表达发生显著改变,其中,上调蛋白10个,最为显著的是ompC基因编码的外膜孔蛋白;下调表达糖蛋白9个,最为显著的是yjgF基因编码的翻译起始抑制因子。【结论】根据定量蛋白质组学分析,沙门氏菌在高盐环境下有多种重要糖蛋白的表达水平发生显著改变,这一结果对研究沙门氏菌SPI-1产生的效应糖蛋白表达情况以及寻找沙门氏菌致病机制有重要意义。     

鼠伤寒沙门氏菌参与细菌竞争相关基因

【背景】细菌在环境中以复杂的微生物群落形式存在,细菌间的竞争是细菌生存的一种重要方式。鼠伤寒沙门氏菌是一种可引起胃肠道疾病的重要人畜共患病病原体,其在水源、食物或是宿主肠道等环境中均需与其他细菌进行相互作用以获得生存优势。【目的】通过转座子技术构建鼠伤寒沙门氏菌转座子插入突变体库,从中筛选鼠伤寒沙门氏菌与细菌竞争能力相关的基因,探讨该菌参与细菌竞争的相关机制。【方法】利用EZ-Tn5^TMTnp Transposome^TM试剂盒获得了含有1 323个突变株的鼠伤寒沙门氏菌突变库,并针对其与大肠杆菌JM109以及MG1655的竞争作用进行筛选,利用反向PCR对筛选出的在细菌竞争能力上具有显著差异的突变株进行侧翼序列的鉴定,确定了转座子插入位点,并根据插入位点所表达基因的功能分析了其造成细菌竞争能力差异的可能原因。【结果】筛选出细菌竞争能力差异显著的13株突变株,其中2株突变株竞争能力显著增强,插入突变的基因为polB与flhd;11株竞争能力显著下降的细菌突变的基因分别为fstJ、rfbG、recC、rfaI、rfaG、rfbC、udha、plsc、mdh、res及ackA。【结论】毒力因子、细菌膜蛋白的完整、细菌代谢能力的正常及其天然免疫能力和DNA的合适修饰等都与细菌参与竞争的能力密切相关,此研究为进一步探讨影响细菌竞争能力的具体机制奠定了基础。     

鼠伤寒沙门氏菌Ⅲ型分泌系统抗感染类抑制剂的筛选

【背景】肠道沙门氏菌(Salmonella enterica)是一种常见的食源性肠道致病菌,可以感染人畜并引发食物中毒、伤寒等疾病。近年来因抗生素滥用导致肠道沙门氏菌耐药性问题日益严峻,迫切需要开发新型抗感染药物。肠道沙门氏菌致病的关键在于与宿主细胞接触后可以通过Ⅲ型分泌系统(type Ⅲ secretion system, T3SS)向宿主细胞内注射效应蛋白,进而调控宿主细胞囊泡运输和免疫应答等生理活动,以方便其高效侵染宿主细胞。T3SS是一类由超过20种蛋白质组成、高度复杂的跨膜分子机器,是革兰氏阴性病原菌中普遍存在的一类蛋白质运输系统和毒力系统。在不同病原菌中,其结构与功能非常保守。位于T3SS核心跨膜区的SctV家族蛋白是T3SS中最保守的组分之一,参与T3SS能量供应和效应蛋白的分泌过程,SctV蛋白的关键氨基酸突变失活后会导致鼠伤寒沙门氏菌丧失对宿主的入侵能力。【目的】以沙门氏菌SctV家族蛋白为靶点,尝试通过虚拟筛选技术筛选与SctV胞内区相互作用的抗感染类T3SS抑制剂。【方法】结合体外相互作用分析、细菌生长曲线实验、细菌分泌实验和细胞侵染实验等对候选分子进行抑制效果的...     

AI-2通过调节c-di-GMP代谢酶DosC影响类志贺邻单胞菌生物膜形成及运动性

细菌中广泛分布的群体感应信号分子autoinducer-2 (AI-2)会影响细菌的生物膜形成及运动性等生理过程,然而该信号对类志贺邻单胞菌相关表型的调控作用及其分子机制尚未有所报道。【目的】揭示AI-2通过影响胞内环二鸟苷单磷酸(c-di-GMP)水平调控类志贺邻单胞菌生物膜形成及运动性的内在机制,为类志贺邻单胞菌感染的防治提供新思路。【方法】首先利用同源重组方法构建luxS基因敲除菌株(ΔluxS),通过软琼脂平板法和结晶紫染色法分别检测其与野生型泳动能力和生物膜形成水平的差异;之后通过序列比对找到AI-2的潜在受体蛋白DosC(SAMEA2665130＿2180),利用哈维氏弧菌生物发光实验及等温滴定量热实验(ITC)研究DosC的配体结合结构域(ligand-binding domain,LBD)与AI-2的结合能力;通过体外酶活实验、胞内c-di-GMP定量分析研究AI-2对DosC受体活性的影响;最后参照前述方法构建受体DosC编码基因敲除菌株(ΔdosC)并检测其与野生型相比泳动能力和生物膜形成水平的变化。【结果】AI-2与DosC-LBD显示出高亲和作用力;通过高效液相...     

The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria

The ubiquitous bacterial second messenger c-di-GMP controls exopolysaccharide synthesis, flagella- and pili-based motility, gene expression, and interactions of bacteria with eukaryotic hosts. With the exception of bacterial cellulose synthases, the identities of c-di-GMP receptors and end targets have remained unknown. Recently, Amikam and Galperin (Amikam, D., and Galperin, M. (2006) Bioinformatics 22, 3-6) hypothesized that the PilZ domains present in the BcsA subunits of bacterial cellulose synthases function in c-di-GMP binding. This hypothesis has been tested here using the Escherichia coli PilZ domain protein YcgR, its individual PilZ domain and the PilZ domain from Gluconacetobacter xylinus BcsA. YcgR was purified and found to bind c-di-GMP tightly and specifically, Kd 0.84 microm. Individual PilZ domains from YcgR and BcsA also bound c-di-GMP, albeit with lesser affinity, indicating that PilZ is sufficient for binding. The site-directed mutagenesis performed on YcgR implicated the most conserved residues in the PilZ domain directly in c-di-GMP binding. It is suggested that c-di-GMP binding to PilZ brings about conformational changes in the protein that stabilize the bound ligand and initiate the downstream signal transduction cascade. While the identity of the downstream partner(s) of YcgR remains unknown, it is shown that YcgR regulates flagellum-based motility in a c-di-GMP-dependent manner. The inactivation of ycgR improves swimming and swarming motility of the poorly motile yhjH mutants of Salmonella enterica serovar Typhimurium UMR1. Therefore, biochemical and genetic evidence presented here establishes PilZ as a long sought after c-di-GMP-binding domain and YcgR as a c-di-GMP receptor affecting motility in enterobacteria.     

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli.

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambda gtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambda Tn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [14C]fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [14C]fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.     

Overproduction of the MotA protein of Escherichia coli and estimation of its wild-type level.

The motA gene of Escherichia coli was placed under the control of a high-level promoter, that of the tryptophan operon of Serratia marcescens. In the presence of the inducer beta-indoleacrylic acid, MotA was synthesized at greatly elevated levels and inserted without apparent limit into the inner membrane. Growth and motility were impaired, but not drastically so, indicating that MotA by itself does not act as a proton ionophore. Antibody raised against the overproduced protein was used to estimate that a wild-type cell contained 600 +/- 250 copies of MotA. This number is more than would be needed to surround each flagellar basal body with a single circlet of MotA protein; possible interpretations of the result are discussed. The antibody was also used to establish that the MotA protein of Salmonella typhimurium has a similar molecular weight to that of E. coli and is immunologically cross-reactive with it; functional complementation of S. typhimurium motA mutants by the E. coli gene was established.     

The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes.

The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.     

Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA.

In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.     

Properties of the galactose binding protein of Salmonella typhimurium and Escherichia coli.

The galactose binding protein implicated in transport and in chemotaxis has been purified to homogeneity from the shock fluids of Salmonella typhimurium and Escherichia coli. Both proteins are monomers of molecular weight 33 000 and exhibit cross-reactivity with antibody. The Salmonella galactose receptor showed binding of 1 mol of [14C]galactose or 1 mol of [14C]glucose at saturation. The dissociation constants were 0.38 and 0.17 muM, respectively. In light of the previously published report that the E. coli protein contains two binding sites with two different affinities, the binding characteristics of this protein were reexamined. Using highly purified radiolabeled substrate and homogeneous protein, a single binding site and single binding affinity were seen galactose (KD = 0.48 muM) or for glucose (KD = 0.21 muM). The competition between glucose and galactose for the same site is intriguing in view of the competition between ribose and galactose at the receptor level.     

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal ( hlyA_S ) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA_S fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium . In S. typhimurium cultures hIL-6-HlyA_S concentrations entered a plateau at 500 to 600 ng ml⁻¹ culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA_S was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA_S fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore, hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.