E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。     

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。     

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。     

北京棒杆菌天冬氨酸激酶突变体R169H的构建及酶学性质表征

【目的】提高北京棒杆菌(Corynebacterium pekinense)中天冬氨酸激酶(aspartokinase,AK)活力。【方法】利用定点突变技术对AK基因进行突变,并将突变体转入大肠杆菌(Escherichia coli)BL21中异源表达。重组菌经超声破碎后、利用镍柱对AK进行纯化,并经SDS-PAGE和Western blot验证。通过检测酶活力比较突变体和野生型动力学变化并研究突变体和野生型的部分酶学性质。【结果】成功构建突变体R169H。经验证知,AK分子量为48kDa。突变体R169H的Vmax为226.3 U/mg·s-1,较野生型提高2.3倍。最适反应温度为26℃,与野生型经验值相同;最适反应pH为9.0,较野生型经验值8.0有所提高;在最适温度和pH值下的半衰期为5.5 h,比野生型的4h稳定性要好;代谢产物赖氨酸、苏氨酸和蛋氨酸在低浓度时对AK均具有激活作用。【结论】突变体中R169与E92间氢键消失,能够影响亚基间聚合度,降低酶对底物的亲和力,减弱代谢产物对AK的反馈抑制作用,从而使R169H中AK的Vmax提高2.3倍。     

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。     

通过定点突变提高乳链菌肽对热及pH的稳定性

【目的】通过定点突变技术改变乳链菌肽(nisin)特定位置氨基酸,获得性质改善的nisin突变体,为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上,对M21K nisZ基因第29位丝氨酸密码子进行定点突变;将其克隆至乳酸菌表达载体pMG36e,并在Lactococcus lactis NZ9800中进行表达;双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ(wild-type,WT)相比,双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降,但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同,可抑制革兰氏阴性菌,扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质,有可能得到应用范围更广的nisin品种。     

毕赤酵母表达的嗜热蓝状菌β-葡萄糖苷酶N-糖基化修饰的作用与功能

【目的】研究N-糖基化对来源于嗜热蓝状菌β-葡萄糖苷酶(β-glucosidase,Bgl3A)的酶学性质影响。【方法】采用定点突变技术构建了3个去N-糖基化的突变体T44A、S228A、S299A,并分别在毕赤酵母GS115中表达纯化。【结果】与野生型Bgl3A相比,突变体S228A分泌蛋白产量极低,仅能微量检测到p NPG活性;突变体T44A和S299A的最适pH和最适温度没有改变,分别为4.0和75°C,但二者的T_m值和70°C下的热稳定性都明显优于野生型。以p NPG为底物时,突变体S299A和T44A的催化效率分别降低了14.5%和70.0%;以纤维二糖为底物时,T44A的催化效率基本不变,而S299A的催化效率提高了1.1倍。【结论】Bgl3A不同位点的N-糖基化修饰对酶的分泌和酶学性质的影响具有明显差异。其中,N226位的N-糖基化在维持酶的表达和功能方面至关重要,而去除N297位点的N-糖基化可以提高酶的热稳定性及对纤维二糖的催化效率。     

定点突变提高D-氨甲酰水解酶的可溶性表达

采用定点突变的方法对皮氏伯克霍尔德氏菌(Burkholderia pickettii)来源的D-氨甲酰水解酶(D-carbamoylase,DCase)编码基因的3个位点A18、Y30、K34进行突变,并将获得的突变体基因片段构建入高表达载体pET-28b中,转化E coli BL21( DE3),获得带有组合三突变(A18E/Y30D/K34E)的DCase-SM表达菌株BL21/pET-DCSM.当以IPTG诱导目的蛋白表达时,发现突变菌株(DCase-SM)与出发菌株(DCase)菌株相比,目的蛋白的可溶性表达显著提高,其可溶蛋白比例约为64％;与出发菌株相比,其单位菌体酶活增加427％;另外,与本实验室前期构建的高可溶性三叠加突变体菌株DCase-M3相比,单位菌体酶活亦增加7.9％.     

霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达

【目的】阐明霍乱弧菌ToxR蛋白功能调控的分子机制。【方法】利用巯基捕获(thiol-trapping)的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用;采用定点突变的方法构建ToxR半胱氨酸突变株(ToxR_(C236/293S));利用荧光素酶基因作为报告基因分析ToxR野生型(ToxR_(wt))和半胱氨酸突变体(ToxR_(C236/293S))诱导下游基因表达的活性;通过细菌双杂交系统分析ToxR_(wt)和ToxR_(C236/293S)蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。【结果】ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化,且当ToxR与ToxS共表达时,ToxR诱导ctxAB转录表达的活性显著增强,且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高;成功构建株霍乱弧菌ToxR半胱氨酸突变株(ToxR_(C236/293S)),在没有ToxS存在的条件下,ToxR_(C236/293S)诱导毒力基因表达的活性与ToxRwt相当;细菌双杂交系统分析发现当ToxR与ToxS共转录表达时,ToxS极大增强ToxR蛋白之间的互作;在dsbA基因缺失突变株中,ToxS之间的相互作用显著增强。【结论】ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响;ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达,而DsbA对ToxS蛋白之间的相互作用具有抑制作用,DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。     

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.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

FtsA forms actin-like protofilaments

FtsA is an early component of the Z‐ring, the structure that divides most bacteria, formed by tubulin‐like FtsZ. FtsA belongs to the actin family of proteins, showing an unusual subdomain architecture. Here we reconstitute the tethering of FtsZ to the membrane via FtsA's C‐terminal amphipathic helix in vitro using Thermotoga maritima proteins. A crystal structure of the FtsA:FtsZ interaction reveals 16 amino acids of the FtsZ tail bound to subdomain 2B of FtsA. The same structure and a second crystal form of FtsA reveal that FtsA forms actin‐like protofilaments with a repeat of 48 Å. The identical repeat is observed when FtsA is polymerized using a lipid monolayer surface and FtsAs from three organisms form polymers in cells when overexpressed, as observed by electron cryotomography. Mutants that disrupt polymerization also show an elongated cell division phenotype in a temperature‐sensitive FtsA background, demonstrating the importance of filament formation for FtsA's function in the Z‐ring.     

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.     

Identification of a region of FtsA required for interaction with FtsZ

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.     

FtsA reshapes membrane architecture and remodels the Z‐ring in Escherichia coli

Cell division in prokaryotes initiates with assembly of the Z‐ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z‐ring is comprised of polymers of tubulin‐like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C‐terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide‐dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C‐terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z‐ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.     

E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments,Reduces GTPase Activity,and Impairs Bacterial Cytokinesis

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7–1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg⁹³-Glu¹³⁸ salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.     

Recruitment of ZipA to the Septal Ring of Escherichia coli Is Dependent on FtsZ and Independent of FtsA

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.     

Oligomerization of FtsZ converts the FtsZ tail motif (conserved carboxy‐terminal peptide) into a multivalent ligand with high avidity for partners ZipA and SlmA

A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (c onserved c arboxy‐t erminal p eptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z‐ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro.