蓝细菌PCC6803染色体上的一对毒素-抗毒素基因的鉴定

大肠杆菌细胞染色体上毒素－抗毒素系统(TA, toxin-antitoxin system)基因参与环境胁迫诱导的细胞死亡或生长抑制。在蓝细菌PCC6803染色体上开放阅读框ssr1114和slr0664具有与TA系统相同的遗传结构, slr0664编码产物与RelBE毒素－抗毒素系统中的毒素蛋白RelE同源, 但没有发现有与ssr1114编码产物同源的蛋白。为证明slr0664和ssr1114表达产物的毒性和抗毒性作用, 构建了含有乳糖诱导调控的启动子和阿拉伯糖诱导调控的启动子的表达调控系统, 将slr0664基因编码序列置于Plac启动子控制下, ssr1114编码序列置于PBAD启动子控制下, slr0664诱导表达产物对细胞具有毒性作用, ssr1114诱导表达产物具有抗slr0664表达产物的毒性作用。提示slr0664为毒素基因, ssr1114为抗毒素基因, 二者组成一个TA系统, 但其有关特性和功能尚待进一步证明。     

集胞藻PCC6803中S2P同源蛋白Slr0643及Sll0862 金属蛋白酶活性的体外鉴定

【目的】金属蛋白酶S2P在细菌中通过在膜切割转录调控因子、释放δ因子参与胁迫响应是跨膜信号转导的保守机制,但蓝细菌中S2P的功能还未被鉴定,故我们考察集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862的金属蛋白酶活性。【方法】以pET-30b(+)为载体,分别构建重组质粒pF0643和pF0862,在大肠杆菌BL21(CE3)中诱导表达并纯化Slr0643及Sll0862蛋白,以β-酪蛋白为底物检测重组蛋白的酶活性。【结果】体外酶活实验显示重组表达的Slr0643及Sll0862蛋白有内切蛋白酶活性,且其活性受金属螯合剂o-phenanthroline的抑制。体外酶活的鉴定结果为进一步研究Slr0643和Sll0862的体内酶活和生物学功能奠定了基础。【结论】集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862具有金属蛋白酶活性。     

高致病性2型猪链球菌毒素-抗毒素系统SezAT的鉴定与活性研究

【目的】对我国高致病性2型猪链球菌05Z33基因组的89K毒力岛序列进行生物信息学分析,发现存在一对与化脓链球菌Epsilon-zeta(ε-ζ)同源的Ⅱ型毒素-抗毒素系统(Toxin-antitoxin system,TA)——SezAT,推测该系统具有稳定89K毒力岛使其不易丢失的作用。验证SezAT为有活性的TA系统。【方法】对SezAT进行了生物信息学分析;RT-PCR验证SezAT共转录特性;在大肠杆菌中选择性地诱导表达毒素蛋白SezT和抗毒素蛋白SezA;最后通过同源重组技术敲除SezAT系统。【结果】sezAT由同一操纵子控制,SezT可抑制细菌生长,SezA可中和SezT的毒性作用,同源重组成功获得sezT敲除突变株。【结论】证实SezAT为一对有活性的毒素-抗毒素(TA)系统,为进一步研究SezAT可能发挥稳定89K毒力岛的功能,同时获得89K毒力岛缺失突变株并深入认识89K在我国高致病性SS2中的作用奠定了基础。     

异源表达的幽门螺杆菌cag4蛋白有溶菌糖基转移酶活性

摘要【目的】构建融合基因原核表达载体pET-28a- cag4,并表达重组融合蛋白cag4,分析重组融合蛋白的酶活性,为新型抗生素（或是抗菌药物）的研发提供作用靶位。【方法】本研究利用PCR技术从幽门螺杆菌NCTC11637中克隆了cag4基因;经T-A克隆,酶切鉴定,构建了原核表达载体pET-28a- cag4;经测序鉴定正确后,转化进入大肠埃希菌 BL21(DE3)进行异源表达。利用IPTG体外诱导后,经SDS-PAGE和Western Bolt鉴定目的蛋白表达后,采用Ni2＋-NTA柱在变性条件下纯化目的蛋白,并对重组蛋白进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析。【结果】在大肠埃希菌 BL21(DE3)中获得高效表达的重组蛋白; 经SDS-PAGE和Western Bolt鉴定表达后,采用Ni2＋-NTA柱在变性条件下纯化,并进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析,表明目的蛋白具有明显的肽聚糖水解活性; 通过监测浊度下降速率,比较其在不同pH条件下活性的变化,即?A/（min?mg protein）,结果表明,幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。【结论】幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。     

细菌毒素-抗毒素系统的功能

毒素-抗毒素(Toxin-Antitoxin,TA)系统广泛存在于原核生物和古细菌的染色体和质粒中。此系统由2个共表达的基因组成,分别编码稳定的毒素蛋白和易降解的抗毒素,毒素通常发挥毒性作用抑制细菌生长,而抗毒素则可中和毒性,二者相互作用对细菌生长状态起精密调节作用。根据TA的组成和抗毒素的性质,目前已经发现有6型TA,这些TA系统在细菌中发挥的作用一直是近年来学者们研究的热点,文中对细菌TA的功能研究进展进行了综述。     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

抗肉毒毒素A人源单链抗体在酵母细胞中的分泌表达*

将特异肉毒抗毒素基因克隆入载体pPIC9k,G418抗性加压筛选阳性整合克隆,在毕赤酵母细胞GS115中进行分泌表达。获得了稳定分泌表达ScFv的工程菌,SDS-PAGE分析可见,目的蛋白分子量约为26kD,通过放大体积来探索重组抗毒素的诱导表达条件及纯化工艺,结果发现,1%甲醇诱导后72～84h,目的蛋白的表达达到高峰,占酵母培养上清中总蛋白的15%以上,经两步层析纯化,目的蛋白纯度可达95%。竞争活性测定结果表明,重组抗毒素在体外具有良好的活性,可竞争肉毒抗毒素马血清与毒素的特异结合。     

蓝细菌Synechocystis PCC 6803染色体上的基因ssl2138和sll1092构成的毒素-抗毒素系统

摘要:【目的】证明集胞藻(Synechocystis)PCC 6803染色体上的假定基因ssl2138和sll1092构成vapBC家族的毒素-抗毒素系统(toxin-antitoxin system,TA系统)。【方法】以RT-PCR分析ssl2138和sll1092的共转录,以选择性表达系统分析其编码产物对大肠杆菌生长的影响,并通过亲和层析和质谱检测证明编码产物之间的相互作用。【结果】ssl2138和sll1092构成的二元操纵子在正常生长条件下共转录;Sll1092表达抑制大肠杆菌的生长,Ssl2138的同时表达或随后表达可拮抗Sll1092的生长抑制作用;Ssl2138与Sll1092相互作用形成复合体。【结论】位于同一操纵子中的假定基因ssl2138和sll1092构成vapBC家族的TA系统。     

利用原核表达系统制备家蚕非典型嗅觉受体Orco抗原蛋白

【目的】探索建立一种有效制备昆虫非典型嗅觉受体Orco抗原的方法,为Orco蛋白组织定位及功能研究奠定基础。【方法】设计带有BamH I和Hind III酶切位点的引物,采用RT-PCR方法扩增家蚕Bombyx mori Orco第 4-5跨膜区之间的基因片段,将其与原核表达载体pET-28a(+)双酶切处理后进行连接,然后转化大肠杆菌Escherichia coli感受态细胞DH5α,重组质粒再转化大肠杆菌BL21(DE3)菌株,用IPTG诱导表达,用SDS-PAGE检测诱导蛋白,用 HisTrap HP亲和层析对诱导表达的大量蛋白进行纯化。【结果】 在大肠杆菌原核表达系统中,用IPTG诱导获得了与预测蛋白大小相符的目的蛋白,经SDS-PAGE检测发现目的蛋白以包涵体形式表达,在变性条件下经HisTrap HP亲和层析获得大量可用于抗体制备的纯化蛋白。【结论】利用原核表达系统可获得制备家蚕Orco抗体的抗原蛋白。     

假结核耶尔森氏菌中Phd-Doc毒素-抗毒素系统的功能鉴定

【背景】毒素-抗毒素系统在微生物体内广泛存在,在微生物对抗外界不良环境方面发挥重要作用。【目的】以模式细菌假结核耶尔森氏菌(Yersinia pseudotuberculosis,Yptb)为材料,探究其编码的Phd-Doc毒素-抗毒素系统的作用机制和生物学功能。【方法】通过生物信息学方法预测Yptb中编码的Phd-Doc毒素-抗毒素系统,通过毒性分析、基因表达分析及蛋白相互作用对其进行鉴定;通过抗生素胁迫、氧胁迫、生物被膜形成等实验研究Phd-Doc在体内发挥的生物学功能。【结果】生物信息学分析鉴定出一对Phd-Doc毒素-抗毒素系统,发现二者共转录且相互作用;毒素蛋白Doc能够引起大肠杆菌形态发生变化并抑制其生长,抗毒素蛋白Phd能中和Doc的毒性;Phd-Doc毒素-抗毒素系统具有自调控抑制效应;phd-doc的缺失对Yptb自身的生长无影响,而且毒素蛋白Doc在野生型Yptb内过表达并未显示毒性;phd-doc在转录水平上响应了抗生素胁迫和氧胁迫,其中,对氯霉素胁迫最为敏感,但并不影响Yptb的生长;同时,Phd-Doc能够影响Yptb的生物被膜形成能力。【结论】Yptb中Phd-Doc毒素-抗毒素系统的功能鉴定对于更好地了解在多变的外部环境下微生物的定殖和响应机制具有重要意义。     

The Proteolytic Activation of the <Emphasis Type="Italic">relNEs</Emphasis> (<Emphasis Type="Italic">ssr1114/slr0664</Emphasis>) Toxin–Antitoxin System by Both Proteases Lons and ClpP2s/Xs of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The proteolytic regulation of toxin–antitoxin (TA) systems has been well studied in Escherichia coli but remains unclear in other bacteria. A chromosomal gene pair ssr1114/slr0664, named relNEs, of Synechocystis sp. PCC 6803 forms a TA system belonging to rel family. Here, we used E. coli strain BL21 (DE3) as a host to characterize the proteolytic regulation of relNEs. The proteases of this strain could not degrade the antitoxin RelN, and the ectopic production of the ATP-dependant protease Lons or ClpP2s/Xs of Synechocystis sp. PCC6803 did not affect E. coli growth. Either Lons or ClpP2s/Xs was able to degrade RelN resulting in growth arrest of E. coli due to the activation of RelEs’s toxicity, and the presence of RelEs could protect RelN to a certain extent against Lons and ClpP2s/Xs. Our observations suggest that both Lons and ClpP2s/Xs are responsible for RelN proteolysis in the native host under certain conditions. RelN is the first protein substrate identified for cyanobacterial ATP-dependent proteases.     

集胞藻PCC 6803类铁氧还蛋白Slr1205的功能研究

类铁氧还蛋白 (ferredoxin-like, Fd-like) 在高等植物中具有调控叶绿体发育等多种重要的生理功能,但在蓝藻中的生物功能尚未被发现。通过比较集胞藻PCC 6083编码Fd-like蛋白基因的敲除突变株Δslr1205与野生型 (WT) 在不同碳源和光周期条件下的生理生化表型,分析Slr1205在集胞藻中的功能。结果显示,在高CO2浓度自养、混合营养和光异养时,Δslr1205的生长速率低于WT,而在空气中自养条件下并无差异。与此相对应,混合营养和光异养时Δslr1205比WT的呼吸速率低,与呼吸作用密切相关的NDH-1L复合体的含量少。Δslr1205在所有测试的条件下有较高的类胡萝卜素以及偏黄的表型。这些数据表明,Fd-like蛋白Slr1205的缺失造成在碳源充足条件下的生长速率下降,这可能是由于呼吸作用下调导致供能不足。研究结果为今后深入研究蓝藻Fd-like蛋白奠定了基础,为开展光合作用和呼吸作用的调节机制研究探索了新方向。     

Slr1122影响Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的色素合成

Synechocystis sp.PCC 6803是一种良好的研究光合作用的模式生物,其中slr1122编码一个250个氨基酸的未知蛋白。据报道Slr1122可能与杂合传感激酶（hybrid sensory kinase）Sll1672（Hik12）相互作用,本研究通过复合物实验证实了Slr1122与Sll1672确实存在相互作用。利用32P标记证明,在加入Slr1122后Hik12的磷酸化受到了明显的影响,推测其可能参与该双组分系统的调控。通过同源双交换,用卡那霉素抗性基因替换slr1122,将slr1122从Synechocystis sp.PCC 6803中敲除,构建了slr1122的缺失体Δslr1122。研究发现在Δslr1122中,编码PSⅡ中核心蛋白D1亚基的slr1181（psbAI）的转录水平明显降低,使PSⅡ光合作用受到影响,导致Δslr1122的生长速率低于野生型（WT）。同时slr1122的缺失使得蓝细菌对光的敏感性增强,在弱光条件下,Δslr1122对光能的利用效率高于WT,其生长速率也较WT高,但与此相反,Δslr1122对强光的耐受力及生长速率则不及WT。Δslr1122体内的藻胆蛋白含量与色素含量均降低,尤其是类胡萝卜素,RT-PCR的结果也显示合成类胡萝卜素过程中的5个关键酶转录水平均下降。这可能是Δslr1122对氧化胁迫变得敏感的原因之一。总之,Slr1122影响杂合传感激酶Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的光合色素合成。     

Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding.

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.     

Slr0077 of Synechocystis has cysteine desulfurase as well as cystine lyase activity

NifS-like proteins activate sulfur for a variety of biosynthetic purposes. The genome of the cyanobacterium Synechocystis contains 4 nifS-related sequences of which only the slr0077 gene seems to be essential. In this report the heterologous production of the Slr0077 protein, its purification, and catalytic properties are described. Slr0077 produces alanine as well as pyruvate from cyst(e)ine as substrate; the product ratio depends on the redox conditions. Alanine is the typical product of orthodox NifS proteins, pyruvate formation is typical of the cystine lyase of Synechocystis which is the most peculiar member of the NifS protein family. The specific activities of Slr0077 for both reaction types are low as compared to the prototypic enzymes. Upon reaction with thiol-alkylating agents Slr0077 is not readily inactivated unlike NifS. The unique properties of Slr0077 add to the emerging picture that the NifS family of proteins comprises enzymes with a variety of distinct reactivities.     

The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen

Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.