FtsZ(236-245)区域的两性螺旋特性对大肠埃希氏菌FtsZ组装和功能的影响

【目的】探索大肠埃希氏菌(Escherichia coli,E.coli)FtsZ(236-245)结构域两性螺旋特性对FtsZ组装和FtsZ-FtsA相互作用的影响。【方法】利用分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化获得相应目标蛋白;通过同源重组和Pl转导构建QN23-QN29菌株;利用活细胞成像观察FtsZ及其突变体的胞内定位特点;膜蛋白分离和Western blot分析FtsZ突变体的膜结合特性变化;非变性胶分离和体外聚合分析检测定点突变对FtsZ单体组装特性的影响;免疫沉淀和Far Western blot实验检测FtsZ/FtsZ~*-FtsA间的相互作用。【结果】FtsZ~(E234A/K)和FtsZ~(E241A/K)突变体的功能活性降低、备突变体在E.coli内不能正确定位和形成功能性Z环;E237A/K和E241A/K位点突变致备突变体聚合能力降低、FtsZ*-FtsA的相互作用减弱和FtsZ的膜结合特性变化。【结论】E237和E241是影响FtsZ(236-245)区域两性螺旋特性和FtsZ组装及FtsZ-FtsA相互作用的重要氨基酸。     

前纤维蛋白点突变对粗糙脉孢菌生长的影响及其生化机制研究

【目的】为进一步了解前纤维蛋白(profilin,PFN)在丝状真菌中的功能,本文以粗糙脉孢菌(Neurospora crassa)为研究对象,进行了前纤维蛋白对其菌落生长和肌动蛋白(actin)聚合特性影响的探究。【方法】通过采用定点突变、同源重组、分生孢子过膜和PCR等技术,获得粗糙脉孢菌前纤维蛋白F78 (F78A和F78D)和V113 (V113E、V113R和V113W)的点突变体。利用平板生长法、竞争性生长管和显微镜观察检测表型变化,并结合多聚脯氨酸亲和层析纯化、荧光分光光度技术和高速共沉淀等技术分析点突变的前纤维蛋白对肌动蛋白聚合特性的影响。【结果】获得的粗糙脉孢菌前纤维蛋白点突变株F78A、F78D、V113E、V113R和V113W,与对照菌株ku70RIP相比,突变株生长均明显减慢(P<0.05),其中PFN (F78D)和PFN (V113W)的突变体在生长的12–48 h,菌落直径分别仅为对照的20.0%–75.7%和12.7%–39.2%。竞争性生长管分析表明,PFN (F78D)和PFN(V113W)突变株菌丝生长速度受到显著抑制,分生孢子形成的节律并...     

Arg77和Glu80定点突变同时去除葡激酶中的T和B细胞抗原表位

【目的】对葡激酶的T和B细胞抗原表位重叠的关键氨基酸Arg77和Glu80进行定点突变以降低葡激酶的免疫原性。【方法】基于Arg77和Glu80的溶剂可及表面积设计葡激酶的突变体;突变体在大肠杆菌DH5α中进行表达。经过三步层析法纯化后,分析突变体的纤溶活性和免疫原性。【结果】免疫学实验提示,葡激酶导致Th2免疫反应;Glu80突变为丙氨酸和丝氨酸减少了溶剂可及表面积,同时去除了部分T和B细胞抗原表位;Arg77突变为天冬酰胺、谷氨酰胺和赖氨酸仅去除了部分T细胞抗原表位;6个组合突变体中,Sak(R77Q/E80A)和Sak(R77Q/E80S)有效去除了部分B和T细胞抗原表位,降低了葡激酶的免疫原性;Sak(R77Q/E80A)and Sak(R77Q/E80S)的纤溶活性和催化效率与r-Sak相当。     

北京棒杆菌AS1.299高丝氨酸脱氢酶突变体L200F/D215K的异源表达及酶学性质

【目的】对北京棒杆菌Corynebacterium pekinense高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)进行空间结构改造从而获得优良性能新酶。【方法】利用定点突变技术构建HSD双突变体L200F/D215A、L200F/D215E、L200F/D215G和L200F/D215K,并将其转入大肠杆菌E.coli BL21中进行高效表达,选取催化效率最高的双突变体L200F/D215K与双突变前的L200F进行动力学和酶学性质比较。【结果】HSD双突变体L200F/D215K的Vmax为36.92 U/mg,较L200F提高1.24倍;最适反应温度为37℃,较L200F提高2℃;最适反应pH为7.5,与L200F经验值相同;最适温度下的半衰期为4.16 h,较L200F提高1.12倍;L200F/D215K和L200F对有机溶剂和金属离子均表现出较好的抗性。【结论】HSD通过空间结构改造活力得到提高,并且其酶学性质得到优化。本研究有助于认识HSD突变体的酶学性质,为其新酶的研发利用提供有力依据。     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

定向引入N-糖基化位点改进黑曲霉β-1,4-内切木聚糖酶热稳定性

【背景】木聚糖是生物圈中仅次于纤维素的第二大多糖,其结构复杂,完全降解需要多种木聚糖酶协同作用。β-1,4-内切木聚糖酶是木聚糖主链水解过程中最关键的酶,已广泛应用于饲料、造纸、能源、食品和医药等行业。但在实际应用中,由于真菌木聚糖酶的热稳定性较差,限制了其在工业中的应用。【目的】提高来源于黑曲霉(Aspergillusniger)的β-1,4-内切木聚糖酶(xynB)热稳定性。【方法】采用氨基酸虚拟突变技术对xynB定向引入一个N-糖基化位点,将虚拟突变后筛选获得的候选突变体和野生型在毕赤酵母SMD1168中表达,并对纯化后的野生型和突变体酶进行酶学性质和稳定性分析。【结果】经虚拟突变和筛选获得5个候选突变体,在毕赤酵母SMD1168中成功表达了4个突变体,其中3个突变体发生了糖基化。突变体和野生型酶均表现出宽范围的酸碱耐受性,且突变体xynB~(A92N/D94T)在pH4.0–11.0条件下的稳定性明显优于野生型;糖基化突变体xynB~(A92N/D94T)、xynB~(G66N/A68T)和xynB~(G66F/D67N/G69T)在温度为60–80°C时热稳定性明显高于野生型,xynB~(G66N/A68T)在80°C保温30 min后的残留酶活比野生型提高了约30%。【结论】本研究方法可为其他来源木聚糖酶和其他工业酶的热稳定分子改造提供参考。     

β-甘露聚糖酶AuMan5A/Af酶学性质的改善与其Asp320的相关性分析

【目的】为改善宇佐美曲霉5家族β-甘露聚糖酶(AuMan5A)的酶学性质,本实验室前期将AuMan5A底物结合凹槽内一个7肽(~(316)KSPDGGN~(322))组成的loop替换为烟曲霉5家族β-甘露聚糖酶对应的氨基酸片段(PSPNDHF),得到loop替换突变酶AuMan5A/Af。为揭示AuMan5A/Af酶学性质显著改善与其Asp~(320)的相关性,定点突变构建突变体AuMan5A/Af~(D320G)。【方法】采用大引物PCR技术将AuMan5A/Af基因(Auman5A/Af)中编码Asp~(320)的密码子GAC突变为Gly~(320)的GGT,构建出突变体基因Auman5A/Af~(D320G),并在毕赤酵母GS115中进行表达,分析表达产物AuMan5A/Af~(D320G)的酶学性质。【结果】AuMan5A/Af~(D320G)的最适温度T_(opt)为70.0℃,变性温度T_m为71.5℃,介于AuMan5A(T_(opt)=65.0℃,T_m=64.5℃)和AuMan5A/Af(T_(opt)=75.0℃,T_m=76.6℃)之间;在70.0℃的半衰期为40 min,高于AuMan5A的10 min,但较AuMan5A/Af的480 min显著缩短;比活性分别是AuMan5A和AuMan5A/Af的2.7和0.3倍;催化效率(k_(cat)/K_m)分别是AuMan5A和AuMan5A/Af的3.9和0.3倍。【结论】将Asp~(320)突变为Gly~(320)显著影响了AuMan5A/Af的酶学性质,证明了Asp~(320)对AuMan5A/Af温度特性改善、比活性和催化效率显著提高的重要作用。     

VP3 G–H环中氨基酸突变对O型口蹄疫病毒生物学特性的影响

【目的】为了研究O型口蹄疫病毒VP3G–H环中氨基酸突变对其生物学特性的影响。【方法】借助口蹄疫病毒反向遗传操作技术平台拯救出2株定点突变体rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)。进行蚀斑形成试验、一步生长曲线的绘制、TCID_(50)和LD_(50)的测定、间接免疫荧光与激光共聚焦显微镜检测。【结果】结果显示,与骨架病毒rHN相比,虽然rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对BHK-21细胞的感染性及其蚀斑表型和复制动力学无显著性差异;但rHN~(V3174Y)和rHN~(D3173N+V3174E+N3179C)对乳鼠的致病力明显减弱,且均获得了小窝蛋白介导侵染CHO-K1细胞的能力。【结论】VP3上第3174位特征性氨基酸突变影响O型口蹄疫病毒感染宿主细胞的毒力及其内吞作用路径,这有助于我们认知VP3 G–H环在口蹄疫病毒粒子立体空间构象中潜在的作用。     

Tyr78-loop对鱼腥藻来源苯丙氨酸脱氨酶活性的影响

【背景】MIO(Methylidene-imidazol-5-one)依赖型酶中,催化因子Tyr所在Loop(Tyr78-loop)的灵活性显著影响酶学性质。【目的】探讨Tyr78-loop对鱼腥藻来源苯丙氨酸脱氨酶酶活的影响,以提高其反应活性。【方法】将该酶的Tyr78-loop进行分子改造,筛选出酶活提高的突变体,并对突变体的酶学性质进行研究。【结果】突变体S73N、E95V、E95K和S73N/E95K在37°C、pH 8.5下比活分别比原酶提高了34%、30%、18%和35%。蛋白三维结构模拟推测在突变体S73N、E95V和E95K中,位于α螺旋与Tyr78-loop交界处的Asn73、Val95和Lys95与附近氨基酸的氢键作用力数目减少,一定程度上增加了Tyr78-loop的柔性。【结论】Ser73位和Glu95位氨基酸的突变增加了Tyr78-loop的灵活性,提高了苯丙氨酸脱氨酶的酶活。     

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto‐ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto‐ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZ_L169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZ_L169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain‐of‐function mutants such as FtsA* or increased levels of the FtsA‐interacting protein FtsN. Similar to FtsA*, FtsZ_L169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZ_L169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.     

Site-specific mutations of FtsZ - effects on GTPase and in vitro assembly

Background  

FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli.     

Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation

An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ⁺ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.     

The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration

The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.     

Examination of the interaction between FtsZ and MinCN in E. coli suggests how MinC disrupts Z rings

In Escherichia coli the Min system prevents Z ring assembly at cell poles by topologically regulating the division inhibitor MinC. The MinC protein has two domains of equal size and both domains can target FtsZ and block cell division in the proper context. Recently, we have shown that, along with MinD, the C‐terminal domain of MinC (MinC^C) competes with FtsA, and to a lesser extent with ZipA, for interaction with the C‐terminal tail of FtsZ to block division. Here we explored the interaction between the N‐terminal domain of MinC (MinC^N) and FtsZ. A search for mutations in ftsZ that confer resistance to MinC^N identified an α‐helix at the interface of FtsZ subunits as being critical for the activity of MinC^N. Focusing on one such mutant FtsZ–N280D, we showed that it greatly reduced the FtsZ–MinC interaction and was resistant to MinC^N both in vivo and in vitro. With these results, an updated model for the action of MinC on FtsZ is proposed: MinC interacts with FtsZ to disrupt two interactions, FtsZ–FtsA/ZipA and FtsZ–FtsZ, both of which are essential for Z ring formation.     

Characterization of ftsZ, the Cell Division Gene of Buchnera aphidicola (Endosymbiont of Aphids) and Detection of the Product

Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division. With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont. Nucleotide sequence determination of a 6.4-kilobase B. aphidicola DNA fragment has indicated that, as in E. coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC–ddlB–ftsA–ftsZ). Although B. aphidicola ftsZ is expressed in E. coli, it cannot complement E. coli ftsZ mutants. High levels of B. aphidicola FtsZ results in the formation of long filamentous E. coli cells, suggesting that this protein interferes with cell division. The presence of FtsZ indicates that in this, as well as in many other previously described properties, B. aphidicola resembles free-living bacteria. Received: 22 July 1997 / Accepted: 28 July 1997     

Suppression of filamentation in recombinant Escherichia coli by amplified FtsZ activity

A stable plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was constructed. Cell filamentation previously observed during the synthesis of poly(3-hydroxybutyric acid), PHB, could be suppressed by the amplified activity of FtsZ. In a defined medium XL1-Blue (pSYL107) accumulated twice as much PHB than XL1-Blue harboring pSYL105, which does not contain the ftsZ gene.     

鸭疫里默氏杆菌流行菌株的分离鉴定及生物学特性

【背景】鸭疫里默氏杆菌(Riemerella anatipestifer,RA)是引起雏鸭、鹅、火鸡等多种家禽、野禽发生传染性浆膜炎的病原,在全世界范围内广泛存在,危害养禽业发展,造成严重的经济损失。【目的】了解国内RA的流行现状,探究该菌的生物学特性,更好地指导防控鸭疫里默氏杆菌病。【方法】对2020-2021年从山东省、河北省、广东省、山西省等地分离的RA疑似菌株进行PCR、生化和血清型鉴定,并根据药物敏感性结果分析耐药现象,根据半数致死量(median lethal dose,LD₅₀)测定结果分析致病力差异。【结果】共鉴定78株RA分离株,其中,血清1型4株、2型21株、10型11株、6型3株、7型17株,1株有交叉凝集现象,21株血清型未定型;药敏结果显示78株分离株对多粘菌素B、磷霉素、克林霉素等的耐药性最强,对头孢拉定、多西环素、呋喃唑酮、氟苯尼考等抗生素最为敏感,并且78株分离株均存在多重耐药性,其中77株耐药5重及以上;动物试验结果显示,RA的分离株致病力普遍较强,而且不同地区分离株、同地区不同分离株之间均存在差异,数株RA分离株的LD₅₀在10⁴-10⁷ CFU不等。【结论】RA在国内流行表现了强致病力及严重耐药情况,本研究结果为更好地预防控制、临床用药及后续致病机制研究提供了参考依据。